Dna sequencing by synthesis using raman and infrared spectroscopy detection

ABSTRACT

This invention provides nucleoside triphosphate analogues having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, wherein R″ is an OH or an H, and wherein R′ is azidomethyl, a hydrocarbyl, or a substituted hydrocarbyl, and which has a Raman spectroscopy peak with wavenumber from 2000 cm −1  to 2300 cm −1  or a Fourier transform-infrared spectroscopy spectroscopy peak with wavenumber from 2000 cm −1  to 2300 cm −1 , and also to methods of DNA sequencing and SNP detection.

This application claims priority of U.S. Provisional Application No.61/489,191, filed May 23, 2011, the content of which is herebyincorporated by reference in its entirety.

This invention was made with government support under grant no. R01HG003582 from the National Institutes of Health. The U.S. Government hascertain rights in this invention.

Throughout this application, certain publications are referenced inparentheses. Full citations for these publications may be foundimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention relates.

BACKGROUND OF THE INVENTION

Sequencing by Synthesis (SBS) has driven much of the “next generation”sequencing technology, allowing the field to approach the $100,000Genome (1-4). With further improvements in nucleotide incorporationdetection methods, SBS could be an engine that drives third-generationplatforms leading to the reality of the “$1,000 Genome”. At the sametime, since non-fluorescent detection approaches are likely to decreasethe cost of obtaining data by avoiding expensive cameras and imagingtools, SBS also offers the possibility of high sensitivity, leading toboth longer reads and permitting single molecule sequencing, therebyremoving one of the most time-consuming and biased steps, the generationand amplification of DNA templates.

Some commercial platforms have been able to achieve direct singlemolecule sequencing but at the expense of accuracy (e.g., a singlefluorescent tag in the case of the Helicoscope tSMS™ technology, 4different fluorophores in Pacific Biosciences' SMRT sequencing approach,or illumination of 4 different fluors by enzyme-attached quantum dots inLife Technologies SMS system) (5-7). The shortcoming in all theseapproaches is that their dependence upon precise timing of a “virtual”pause between each nucleotide incorporation event, especially whenregistering the incorporation of more than a single base. This becomesparticularly pronounced with homopolymeric runs of more than about 4bases, which are often resolved by summing the fluorescent signals,rather than attempting to measure their timing (8,9). The use ofreversible terminators overcomes this obstacle by only allowing a singlebase to be incorporated prior to the detection step; only aftersubsequent cleavage of the terminating moiety on the nucleotide, can thenext one be incorporated and identified (10-13). In the case of analready established system with fluorescently tagged nucleotidereversible terminators (NRTs), because each of the nucleotides has aseparate fluorescent tag, all four can be added at the same time,reducing the number of rounds of incorporation 4-fold (14, 15). It isnoteworthy that this strategy has also been shown to solve the accuracyproblem for pyrosequencing, used by a Roche sequencing platform, whichis not a single-molecule approach (16).

SUMMARY OF THE INVENTION

A nucleoside triphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, wherein R″ is OH or H, and wherein R′ is azidomethyl, ahydrocarbyl, or a substituted hydrocarbyl, which (i) preferably has oneof the following structures:

wherein m is C₁-C₅, preferably C₁;

-   -   n is C₁-C₅, preferably C₁; and    -   q is C₁-C₅, preferably C₁; and i is C₀-C₄, preferably C₀.        and (ii) has a Raman spectroscopy peak with wavenumber from 2000        cm⁻¹ to 2300 cm⁻¹ or a Fourier transform-infrared spectroscopy        peak with wavenumber from 2000 cm⁻¹ to 2300 cm⁻¹.

In one embodiment, the nucleoside triphosphate analogue R″ is H and thenucleoside triphosphate analogue is a deoxyribonucleoside triphosphateanalogue. In another embodiment, the nucleoside triphosphate analogue R″is OH and the nucleoside triphosphate analogue is a ribonucleosidetriphosphate analogue.

This invention also provides a polynucleotide analogue, wherein thepolynucleotide analogue differs from a polynucleotide by comprising atits 3′ terminus one of the following structures in place of the H atomof the 3′ OH group of the polynucleotide:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom.

Further, this invention provides a composition comprising fourdeoxyribonucleoside triphosphate (dNTP) analogues, each dNTP analoguehaving the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, and wherein (i) the structure of the R′ group of each of the fourdNTP analogues is different from the structure of the R′ group of theremaining three dNTP analogues, and (ii) each of the four dNTP analoguescomprises a base which is different from the base of the remaining threedNTP analogues.

Still further this invention provides a method for determining thesequence of consecutive nucleotide residues of a single-stranded DNAcomprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof, with a DNA polymerase and four        deoxyribonucleoside triphosphate (dNTP) analogues under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to a nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, so as to form a DNA extension product, wherein (i) each        of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group and (b) has a predetermined            Raman spectroscopy peak with wavenumber of from 2000 cm⁻¹ to            2300 cm⁻¹ and which is different from the wavenumber of the            Raman spectroscopy peak of the other three dNTP analogues,            and (iii) each of the four dNTP analogues comprises a base            which is different from the base of the other three dNTP            analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product;

    -   (c) determining after step (b) the wavenumber of the Raman        spectroscopy peak of the dNTP analogue incorporated in step (a)        so as to thereby determine the identity of the incorporated dNTP        analogue and thus determine the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   (d) treating the incorporated nucleotide analogue under specific        conditions so as to replace the R′ group thereof with an H atom        thereby providing a 3′ OH group at the 3′ terminal of the DNA        extension product; and

    -   (e) iteratively performing steps (a) to (d) for each nucleotide        residue of the single-stranded DNA to be sequenced except that        in each repeat of step (a) the dNTP analogue is (i) incorporated        into the DNA extension product resulting from a preceding        iteration of step (a), and (ii) complementary to a nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product        resulting from a preceding iteration of step (a), so as to form        a subsequent DNA extension product, with the proviso that for        the last nucleotide residue to be sequenced step (d) is        optional,        thereby determining the identity of each of the consecutive        nucleotide residues of the single-stranded DNA so as to thereby        sequence the DNA.

In addition, this invention provides a method for determining thesequence of consecutive nucleotide residues of a single-stranded DNAcomprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof, with a DNA polymerase and four        deoxyribonucleoside triphosphate (dNTP) analogues under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to a nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, so as to form a DNA extension product, wherein (i) each        of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group, and (b) has a predetermined            Fourier transform-infrared spectroscopy peak with wavenumber            of from 2000 cm⁻¹ to 2300 cm⁻¹ and which is different from            the wavenumber of the Fourier transform-infrared            spectroscopy peak of the other three dNTP analogues,            and (iii) each of the four dNTP analogues comprises a base            which is different from the base of the other three dNTP            analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product;

    -   (c) determining after step (b) the wavenumber of the Fourier        transform-infrared spectroscopy peak of the dNTP analogue        incorporated in step (a) so as to thereby determine the identity        of the incorporated dNTP analogue and thus determine the        identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   (d) treating the incorporated nucleotide analogue under specific        conditions so as to replace the R′ group thereof with an H atom        thereby providing a 3′ OH group at the 3′ terminal of the DNA        extension product; and

    -   (e) iteratively performing steps (a) to (d) for each nucleotide        residue of the single-stranded DNA to be sequenced except that        in each repeat of step (a) the dNTP analogue is (i) incorporated        into the DNA extension product resulting from a preceding        iteration of step (a), and (ii) complementary to a nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product        resulting from a preceding iteration of step (a), so as to form        a subsequent DNA extension product, with the proviso that for        the last nucleotide residue to be sequenced step (d) is        optional,        thereby determining the identity of each of the consecutive        nucleotide residues of the single-stranded DNA so as to thereby        sequence the DNA.

Further, this invention provides a composition comprising fourribonucleoside triphosphate (rNTP) analogues, each rNTP analogue havingthe structure:

wherein B is a base and is adenine, guanine, cytosine, or uracil, andwherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, and wherein (i) the structure of the R′ group of each of the fourrNTP analogues is different from the structure of the R′ group of theremaining three rNTP analogues, and (ii) each of the four rNTP analoguescomprises a base which is different from the base of the remaining threerNTP analogues.

This invention also provides a nucleoside triphosphate analogue havingthe structure:

wherein the base is adenine, guanine, cytosine, uracil or thymine,wherein R″ is an OH or an H,

wherein L a cleavable linker, and

wherein R has the structure:

wherein the wavy line indicates the point of attachment to L.

This invention further provides a process for labeling a polynucleotidecomprising: contacting the polynucleotide with a deoxyribonucleosidetriphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom,

in the presence of a DNA polymerase under conditions permittingincorporation of the deoxyribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between thedeoxyribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide.

This invention yet further provides a process for labeling apolynucleotide comprising: contacting the polynucleotide with aribonucleotide triphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, or uracil, andwherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom,

in the presence of an RNA polymerase under conditions permittingincorporation of the ribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between theribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide.

In certain embodiments, this invention provides a method for determiningthe identity of a nucleotide residue within a stretch of consecutivenucleic acid residues in a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof such that the 3′ terminal        nucleotide residue of the primer is hybridized to a nucleotide        residue of the single-stranded DNA immediately 3′ to the        nucleotide residue being identified, with a DNA polymerase and a        least four deoxyribonucleoside triphosphate (dNTP) analogues        under conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to the nucleotide residue of the single-stranded DNA being        identified, so as to form a DNA extension product, wherein (i)        each of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group, and (b) has a predetermined            Raman spectroscopy peak with wavenumber which is from 2000            cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of            the Raman spectroscopy peak of the other three dNTP            analogues or has a predetermined Fourier transform-infrared            spectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300            cm⁻¹ and which is different from the wavenumber of the            Fourier transform-infrared spectroscopy peak of the other            three dNTP analogues, and (iii) each of the four dNTP            analogues comprises a base which is different from the base            of the other three dNTP analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product; and

    -   (c) determining the wavenumber of the Raman spectroscopy peak or        wavenumber of the Fourier transform-infrared spectroscopy peak        of the dNTP analogue incorporated in step (a) so as to thereby        determine the identity of the incorporated dNTP analogue and        thus determine the identity of the complementary nucleotide        residue in the single-stranded DNA,        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

In yet further embodiments this invention provides a method fordetermining the identity of a nucleotide residue within a stretch ofconsecutive nucleic acid residues in a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof such that the 3′ terminal        nucleotide residue of the primer is hybridized to a nucleotide        residue of the single-stranded DNA immediately 3′ to the        nucleotide residue identified, with a DNA polymerase and a        deoxyribonucleoside triphosphate (dNTP) analogue under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of the dNTP analogue if it is        complementary to the nucleotide residue of the single-stranded        DNA being identified, so as to form a DNA extension product,        wherein (i) the dNTP analogue has the structure:

wherein B is a base and is adenine, guanine, cytosine, or thymine, and(ii) the dNTP analogue has an R′ group which is azidomethyl, or asubstituted or unsubstituted hydrocarbyl group and has a predeterminedRaman spectroscopy peak with wavenumber which is from 2000 cm⁻¹ to 2300cm⁻¹ or a predetermined Fourier transform-infrared spectroscopy peakwith wavenumber which is from 2000 cm⁻¹ to 2300 cm⁻¹;

-   -   (b) removing dNTP analogues not incorporated into the DNA        extension product; and    -   (c) determining if the dNTP analogue was incorporated into the        primer in step (a) by measuring after step (b) the wavenumber of        the Raman spectroscopy peak or wavenumber of the Fourier        transform-infrared spectroscopy peak of any dNTP analogue        incorporated in step (a), wherein (1) if the dNTP analogue was        incorporated in step (a) determining from the wavenumber of the        Raman spectroscopy peak or wavenumber of the Fourier        transform-infrared spectroscopy peak measured the identity of        the incorporated dNTP analogue and thus determining the identity        of the complementary nucleotide residue in the single-stranded        DNA, and (2) wherein if the dNTP analogue was not incorporated        in step (a) iteratively performing steps (a) through (c) until        the complementary nucleotide residue in the single-stranded DNA        is identified, with the proviso that each dNTP analogue used to        contact the single-stranded DNA template in each subsequent        iteration of step (a), (i) has a predetermined Raman        spectroscopy peak with wavenumber which is different from the        wavenumber of the Raman spectroscopy peak of every dNTP analogue        used in preceding iterations of step (a) or has a predetermined        Fourier transform-infrared spectroscopy peak which is different        from the Fourier transform-infrared spectroscopy peak of every        dNTP analogue used in preceding iterations of step (a), and (ii)        comprises a base which is different from the base of every dNTP        analogue used in preceding iterations of step (a),        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

In this further embodiment, this invention provides a method fordetermining the identity of a nucleotide residue within a stretch ofconsecutive nucleic acid residues in a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA with four different        oligonucleotide probes, (1) wherein each of the oligonucleotide        probes comprises (i) a portion that is complementary to a        portion of consecutive nucleotides of the single stranded DNA        immediately 3′ to the nucleotide residue being identified,        and (ii) a 3′ terminal nucleotide residue analogue comprising on        its sugar a 3′-O—R′ group wherein R′ is (a) is azidomethyl, or a        substituted or unsubstituted hydrocarbyl group and (b) has a        predetermined Raman spectroscopy peak with wavenumber which is        from 2000 cm⁻¹ to 2300 cm⁻¹, and which is different from the        wavenumber of the Raman spectroscopy peak of the R′ of the 3′        terminal nucleotide residue analogue of the other three        oligonucleotide probes, or has a predetermined Fourier-transform        infra red spectroscopy peak with wavenumber which is from 2000        cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of the        Fourier-transform infra red peak of the R′ of the 3′ terminal        nucleotide residue analogue of the other three oligonucleotide        probes, and (iii) each of the four terminal nucleotide residue        analogue comprises a base which is different from the base of        the terminal nucleotide residue analogue of the other three        oligonucleotide probes, and (2) under conditions permitting        hybridization of the primer which is fully complementary to the        portion of consecutive nucleotides of the single stranded DNA        immediately 3′ to the nucleotide residue being identified;    -   (b) removing oligonucleotide primers not hybridized to the        single-stranded DNA; and    -   (c) determining the wavenumber of the Raman spectroscopy peak or        wavenumber of the Fourier-transform infra red peak of the dNTP        analogue of the oligonucleotide probe hybridized in step (a) so        as to thereby determine the identity of the dNTP analogue of the        hybridized oligonucleotide probe and thus determine the identity        of the complementary nucleotide residue in the single-stranded        DNA,        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Sequencing by synthesis (SBS) with nucleotides reversibleterminators (NRTs). (A) The scheme for 4-color SBS with3′-O—N₃-dNTP-N₃-dye NRTs and unlabeled NRTs. All NRTs, which aresubstituted with azidomethyl groups at the 3′-OH group and 4 of whichhave fluorophores attached via linkers also containing azidomethylgroups, are present together in the polymerase reaction. Followingincorporation of the correct base and its determination by fluorescentscanning, Tris-(2-carboxyethyl)phosphine (TCEP) is added to cleave thedyes and at the same time restore the 3′-OH group for the next reactioncycle. Unlabeled NRTs, 3′-O—N₃-dNTPs (without attached dyes) areincluded in the reaction and incorporated as well between reactions toensure that essentially all the primers have been extended (i.e., tosynchronize the reactions). (B) A typical result on a surface-boundsynthetic template using the 3′-O—N₃-dNTP-N₃-dye NRTs.

FIG. 2. A set of four 3′-O-azidomethyl nucleotide reversible terminatorsthat can be used for SBS with solution and solid substrate Raman andSERS detection.

FIG. 3. Mechanism of 3′-O-azidomethyl cleavage byTris-(2-carboxyethyl)phosphine (TCEP) to regenerate the 3′-OH.

FIG. 4. Experimental scheme of continuous IR-SBS using 3′-azidomethylmodified NRTs (Left) and the Fourier-transform infrared (FTIR) spectra(Right) of the products from each step (light gray, N₃ IR signal on theDNA extension products; dark gray, IR signal of the DNA extensionproducts with the azidomethyl group removed). Only the portion of theFTIR spectrum in the range from 2150 cm⁻¹ to 2050 cm⁻¹ (from left toright) is shown, the azide (N₃) peak appearing at 2115 cm⁻¹.

FIG. 5. Raman spectra of 4 chemical tags from 2100 cm⁻¹ to 2260 cm⁻¹,where DNA and protein have no Raman peaks. The four tags (—N₃, —CN,—C≡H, and —C≡CH₃) with strong Raman peaks at 2105 cm⁻¹ (—N₃), 2138 cm⁻¹(—C≡CH), 2249 cm⁻¹ (—C≡CH₃), 2259 cm⁻¹ (—CN) are used to label A, C, G,and T in the “4-signal” Raman SBS.

FIG. 6. SERS spectra of RH800, showing single molecule SERS events forthe cyano (—C≡N) group.

FIG. 7. Scanning electron micrograph of Au-coated, lithographicallypatterned Klarite substrate.

FIG. 8. Experimental high enhancement factor for azido (—N₃) chemicaltag. (a) Typical Raman spectra of 1 μM solution coated on Klaritesubstrate and the corresponding reference Raman spectra of 100 mMsolution coated on aluminum (b).

FIG. 9. Raman signal of 3′-O—N₃-dNTPs (N₃-dNTPs) (red) and natural dNTPs(blue). In all 4 cases, there is a 10²-fold signal increase at theexpected Raman shift of ˜2125 cm⁻¹ due to the N₃ group.

FIG. 10. Scheme of continuous DNA sequencing by synthesis (middle) usingreversible terminators, 3′-O—N₃-dNTPs, with Raman (left) and MALDI-TOFMS spectra (right) obtained at each step. Only the 2000-2300 cm⁻¹ Ramaninterval is shown, the azide peak appearing at Raman shift ˜2125 cm⁻¹.Pol=DNA polymerase.

FIG. 11. a) Concept for nanoantennae-enhanced SERS: the plasmonic cavityincreases the optical field excitation by more than 100 times, whileenhancing the scattering rate by nearly an order of magnitude. b) SEM ofsub-10 nm gap made by e-beam lithography.

FIG. 12. Spatially resolved coupling between a single nitrogen vacancycolor center and an optical mode of a planar photonic crystalnanocavity, showing the spatially resolved Purcell effect in a solid.

FIG. 13. Process for forming bowtie antenna structures with selectiveplacement of DNA within the gap. a) E-beam lithography and development.b) Deposition of spacer, Ag and Ti, followed by liftoff. c) SiO₂deposition by CVD, followed by HMDS. d) Second lithography to defineadhesion sites for DNA primer. e) Primer assembly.

FIG. 14. Bowtie antenna with DNA polymerase bound to a patterned nanodotcentered in the hotspot.

FIG. 15. Top: Au nanoparticle binding to DNA origami. Many differentconfigurations are possible. Bottom: Lithographically directed placementof origami scaffolds (left: rectangles; right: triangles).

FIG. 16. Left: Plasmonic crystal consisting of a patterned TiO-Au-TiOlayer on InP. Right: Resonant plasmonic mode for coupling to moleculeson the metal surface.

FIG. 17. An overall scheme for SERS-SBS with 3′-O-azidomethyl dNTPs.Surface-attached templates are extended with NRTs, added one at a time.If there is incorporation, a Raman signal (˜2105 cm⁻¹) due to the N₃group is detected. After cleavage of the blocking group with TCEP, thenext cycle is initiated. Because the NRTs force the reactions to pauseafter each cycle, the lengths of homopolymers are determined withprecision.

FIG. 18. Four modified nucleotide reversible terminators with distinct3′-O-Raman tags for use in the design and synthesis of novel NRTs with 4distinct SERS signatures to perform SERS-SBS. Their Raman peaks are alsoindicated.

FIG. 19. Generalized synthetic scheme for NRTs shown in FIG. 7. This isa straightforward modification of the protocol used to produce the3′-O-azidomethyl-NRTs for use in SBS in solution with Raman and SERSdetection. In brief, 5′-protected 2′-deoxynucleosides are treated withdisubstituted ethyl sulfoxide and acetic anhydride/acetic acid toproduce, via a Pummerer rearrangement, intermediates that then reactwith sulfuryl chloride and sodium azide to afford 3′-azidomodifiednucleoside derivatives.

FIG. 20. Mechanism of cleavage of the 3′ group of an incorporatednucleotide analogue (adenine base) as depicted in FIG. 7. A similarscheme operates for all the Raman tags attached to the 3′-OH group. Notethe reversion to the 3′-OH and destruction of the N₃ group.

FIG. 21. Alternative NRTs with distinct Raman tags placed either on thebase via NH₂ groups on A and C, or at the 3′-OH of the sugar for G andT. In the case of the A and C analogs at the top of the figure,treatment with TCEP removes both the substitution on the base and at the3′-OH position at the same time, resulting in restoration of intact dATPand dCTP.

FIG. 22. Synthetic scheme for generation of the C analog substituted viathe amino group on the base with a tag displaying two Raman peaks due tothe presence of both an N₃ and alkyne group.

FIG. 23. Overall design of SERS-SBS for 4 mixed nucleotides with fourdifferent Raman signatures. A specific NRT complementary to the nextbase in the covalently attached template added by polymerase to thehybridized primer is decoded via its Raman signature. After cleavage ofthe 3′-O-tag by TCEP, the next cycle can ensue. Note that all 4 NRTshave an azido peak, but in addition 3 of the NRTs have a seconddiscriminating peak. The Raman signatures are shown in FIG. 5.

FIG. 24. Structures of the nucleotide reversible terminators,3′-O—N₃-dATP, 3′-O—N₃-dTTP, 3′-O—N₃-dGTP, and 3′-O—N₃-dCTP.

FIG. 25. Mechanisms to cleave the 3′-O-azidomethyl group from the DNAextension products with TCEP to regenerate the 3′-OH group.

FIG. 26. Polymerase DNA extension reaction using 3′-O—N₃-dNTPs asreversible terminators.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides nucleoside triphosphate analogue having thestructure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, wherein R″ is OH or H, and wherein R′ is azidomethyl, ahydrocarbyl, or a substituted hydrocarbyl, which (i)

-   -   wherein m is C₁-C₅, preferably C₁;        -   n is C₁-C₅, preferably C₁; and        -   q is C₁-C₅, preferably C₁; and i is C₀-C₄, preferably C₀.            and (ii) has a Raman spectroscopy peak with wavenumber from            2000 cm⁻¹ to 2300 cm⁻¹ or a Fourier transform-infrared            spectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300            cm⁻¹.

In one embodiment, the invention provides a nucleoside triphosphateanalogue wherein R″ is H and the nucleoside triphosphate analogue is adeoxyribonucleoside triphosphate analogue. In another embodiment, theinvention provides a nucleoside triphosphate analogue wherein R″ is OHand the nucleoside triphosphate analogue is a ribonucleosidetriphosphate analogue.

In a further embodiment of the invention the nucleoside triphosphateanalogue R′ has one of the following structures, wherein the wavy lineindicates the point of attachment of R′ to the 3′ O atom:

In certain embodiments of the invention the nucleoside triphosphateanalogue is recognized by a DNA polymerase or by an RNA polymeraseand/or R′ has a Raman spectroscopy peak with wavenumber from 2100 cm⁻¹to 2260 cm⁻¹.

This invention additionally concerns polynucleotide analogue, whereinthe polynucleotide analogue differs from a polynucleotide by comprisingat its 3′ terminus one of the following structures in place of the Hatom of the 3′ OH group of the polynucleotide:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom.

This invention also concerns a composition comprising fourdeoxyribonucleoside triphosphate (dNTP) analogues, each dNTP analoguehaving the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, and wherein (i) the structure of the R′ group of each of the fourdNTP analogues is different from the structure of the R′ group of theremaining three dNTP analogues, and (ii) each of the four dNTP analoguescomprises a base which is different from the base of the remaining threedNTP analogues.

In addition, this invention provides a method for determining thesequence of consecutive nucleotide residues of a single-stranded DNAcomprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof, with a DNA polymerase and four        deoxyribonucleoside triphosphate (dNTP) analogues under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to a nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, so as to form a DNA extension product, wherein (i) each        of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group and (b) has a predetermined            Raman spectroscopy peak with wavenumber of from 2000 cm⁻¹ to            2300 cm⁻¹ and which is different from the wavenumber of the            Raman spectroscopy peak of the other three dNTP analogues,            and (iii) each of the four dNTP analogues comprises a base            which is different from the base of the other three dNTP            analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product;

    -   (c) determining after step (b) the wavenumber of the Raman        spectroscopy peak of the dNTP analogue incorporated in step (a)        so as to thereby determine the identity of the incorporated dNTP        analogue and thus determine the identity of the complementary        nucleotide residue in the single-stranded DNA;

    -   (d) treating the incorporated nucleotide analogue under specific        conditions so as to replace the R′ group thereof with an H atom        thereby providing a 3′ OH group at the 3′ terminal of the DNA        extension product; and

    -   (e) iteratively performing steps (a) to (d) for each nucleotide        residue of the single-stranded DNA to be sequenced except that        in each repeat of step (a) the dNTP analogue is (i) incorporated        into the DNA extension product resulting from a preceding        iteration of step (a), and (ii) complementary to a nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product        resulting from a preceding iteration of step (a), so as to form        a subsequent DNA extension product, with the proviso that for        the last nucleotide residue to be sequenced step (d) is        optional,        thereby determining the identity of each of the consecutive        nucleotide residues of the single-stranded DNA so as to thereby        sequence the DNA.

Further, this invention provides a method for determining the sequenceof consecutive nucleotide residues of a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof, with a DNA polymerase and four        deoxyribonucleoside triphosphate (dNTP) analogues under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to a nucleotide residue of the single-stranded DNA which is        immediately 5′ to a nucleotide residue of the single-stranded        DNA hybridized to the 3′ terminal nucleotide residue of the        primer, so as to form a DNA extension product, wherein (i) each        of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group and (b) has a predetermined            Fourier transform-infrared spectroscopy peak with wavenumber            of from 2000 cm⁻¹ to 2300 cm⁻¹ and which is different from            the wavenumber of the Fourier transform-infrared            spectroscopy peak of the other three dNTP analogues,            and (iii) each of the four dNTP analogues comprises a base            which is different from the base of the other three dNTP            analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product;

    -   (c) determining after step (b) the peak Fourier        transform-infrared spectroscopy wavenumber of the dNTP analogue        incorporated in step (a) so as to thereby determine the identity        of the incorporated dNTP analogue and thus determine the        identity of the complementary nucleotide residue in the        single-stranded DNA;

    -   (d) treating the incorporated nucleotide analogue under specific        conditions so as to replace the R′ group thereof with an H atom        thereby providing a 3′ OH group at the 3′ terminal of the DNA        extension product; and

    -   (e) iteratively performing steps (a) to (d) for each nucleotide        residue of the single-stranded DNA to be sequenced except that        in each repeat of step (a) the dNTP analogue is (i) incorporated        into the DNA extension product resulting from a preceding        iteration of step (a), and (ii) complementary to a nucleotide        residue of the single-stranded DNA which is immediately 5′ to a        nucleotide residue of the single-stranded DNA hybridized to the        3′ terminal nucleotide residue of the DNA extension product        resulting from a preceding iteration of step (a), so as to form        a subsequent DNA extension product, with the proviso that for        the last nucleotide residue to be sequenced step (d) is        optional,        thereby determining the identity of each of the consecutive        nucleotide residues of the single-stranded DNA so as to thereby        sequence the DNA.

In an embodiment of the instant methods the dNTP analogues have a Ramanspectroscopy peak with wavenumber of from 2100 cm⁻¹ to 2260 cm⁻¹. In anembodiment of the instant methods the wavenumber of the Fouriertransform-infrared spectroscopy peak is determined by irradiating theincorporated dNTP analogue with grazing angle infra-red light. In anembodiment of the instant methods the peak Raman spectroscopy wavenumberis determined by irradiating the incorporated dNTP analogue with 532 nm,633 nm, or 785 nm light. In an embodiment of the instant methods atleast one of the primer or the single-stranded DNA is attached to asolid surface. In an embodiment of the instant methods the Ramanspectroscopy is surface-enhanced Raman spectroscopy (SERS). In anembodiment of the process the polynucleotide is attached to a solidsurface. In an embodiment of the process the solid surface is metal oris coated with metal or is impregnated with metal. In an embodiment ofthe process the solid surface is porous alumina impregnated with silveror gold. In an embodiment of the process the porous alumina solidsurface is in the form of a nanotube. In an embodiment of the instantmethods in the dNTP analogues R′ has a structure chosen from thefollowing:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom.

In certain embodiments of the methods of this invention the dNTPanalogues each have the structure:

where R^(x) is, independently, a C1-C5 cyanoalkyl, a C1-C5 alkyl, aC2-C5 alkenyl, or a C2-C5 alkynyl, which is substituted orunsubstituted.

In some embodiment of the methods of this invention, in step (d) theincorporated nucleotide analogue is treated with a specific chemicalagent so as to replace the R′ group thereof with an H atom therebyproviding a 3′ OH group at the 3′ terminal of the DNA extension product.In other embodiments of the methods, in step (d) the incorporatednucleotide analogue is treated with Tris (2-carboxyethyl)phosphine(TCEP) so as to replace the R′ group thereof with an H atom therebyproviding a 3′ OH group at the 3′ terminal of the DNA extension product.

Further this invention concerns a composition comprising fourribonucleoside triphosphate (rNTP) analogues, each rNTP analogue havingthe structure:

wherein B is a base and is adenine, guanine, cytosine, or uracil, andwherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, and wherein (i) the structure of the R′ group of each of the fourrNTP analogues is different from the structure of the R′ group of theremaining three rNTP analogues, and (ii) each of the four rNTP analoguescomprises a base which is different from the base of the remaining threerNTP analogues.

In some embodiments the invention provides a nucleoside triphosphateanalogue having the structure:

wherein the base is adenine, guanine, cytosine, uracil or thymine,

wherein R″ is an OH or an H,

wherein L a cleavable linker, and

wherein R has the structure:

wherein the wavy line indicates the point of attachment to L.

In certain embodiments of the nucleoside triphosphate analogue, L is asingle covalent bond. In some embodiments L comprises one or morephotocleavable covalent bonds. In other embodiment R″ is H and thenucleoside triphosphate analogue is a deoxyribonucleoside triphosphateanalogue. Yet still other embodiments R″ is OH and the nucleosidetriphosphate analogue is a ribonucleoside triphosphate analogue.

This invention also concerns a process for labeling a polynucleotidecomprising: contacting the polynucleotide with a deoxyribonucleosidetriphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom,

in the presence of a DNA polymerase under conditions permittingincorporation of the deoxyribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between thedeoxyribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide.

In certain embodiment of this process, the polynucleotide is adeoxyribonucleic acid. In other embodiment of the process, thedeoxyribonucleic acid is a primer. In still other embodiments of theprocess, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase or 9° N polymerase (exo-)A485L/Y409V.

This invention yet further provides a process for labeling apolynucleotide comprising: contacting the polynucleotide with aribonucleoside triphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, or uracil, andwherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom,

in the presence of an RNA polymerase under conditions permittingincorporation of the ribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between theribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide.

In embodiments of this process, the polynucleotide is a ribonucleicacid. In certain embodiment of the process, the polymerase isBacteriophage SP6, T7 or T3 RNA polymerases. In other embodiment of theprocess, the polynucleotide is attached to a solid surface. In certainembodiment of the process, the solid surface is metal or is coated withmetal or is impregnated with metal. In more specific embodiments of theprocess, the solid surface is porous alumina impregnated with silver orgold; or is a porous alumina solid surface in the form of a nanotube.

In certain embodiment of the process, the single-stranded DNA isamplified from a sample of DNA prior to step (a).

In some embodiments of the process the single-stranded DNA is amplifiedby a polymerase chain reaction.

This invention also concerns a method for determining the identity of anucleotide residue within a stretch of consecutive nucleic acid residuesin a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof such that the 3′ terminal        nucleotide residue of the primer is hybridized to a nucleotide        residue of the single-stranded DNA immediately 3′ to the        nucleotide residue being identified, with a DNA polymerase and a        least four deoxyribonucleoside triphosphate (dNTP) analogues        under conditions permitting the DNA polymerase to catalyze        incorporation into the primer of a dNTP analogue complementary        to the nucleotide residue of the single-stranded DNA being        identified, so as to form a DNA extension product, wherein (i)        each of the four dNTP analogues has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, (ii) each of the four dNTP analogues has an R′            group which (a) is azidomethyl, or a substituted or            unsubstituted hydrocarbyl group and (b) has a predetermined            Raman spectroscopy peak with wavenumber which is from 2000            cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of            the Raman spectroscopy peak of the other three dNTP            analogues or has a predetermined Fourier transform-infrared            spectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300            cm⁻¹ and which is different from the wavenumber of the            Fourier transform-infrared spectroscopy peak of the other            three dNTP analogues, and (iii) each of the four dNTP            analogues comprises a base which is different from the base            of the other three dNTP analogues;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product; and

    -   (c) determining the wavenumber of the Raman spectroscopy peak or        wavenumber of the Fourier transform-infrared spectroscopy peak        of the dNTP analogue incorporated in step (a) so as to thereby        determine the identity of the incorporated dNTP analogue and        thus determine the identity of the complementary nucleotide        residue in the single-stranded DNA,        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

Yet further this invention concerns a method for determining theidentity of a nucleotide residue within a stretch of consecutive nucleicacid residues in a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA, having a primer        hybridized to a portion thereof such that the 3′ terminal        nucleotide residue of the primer is hybridized to a nucleotide        residue of the single-stranded DNA immediately 3′ to the        nucleotide residue to be identified, with a DNA polymerase and a        deoxyribonucleoside triphosphate (dNTP) analogue under        conditions permitting the DNA polymerase to catalyze        incorporation into the primer of the dNTP analogue if it is        complementary to the nucleotide residue of the single-stranded        DNA being identified, so as to form a DNA extension product,        wherein (i) the dNTP analogue has the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine, or            thymine, and (ii) the dNTP analogue has an R′ group which is            azidomethyl, or a substituted or unsubstituted hydrocarbyl            group and has a predetermined Raman spectroscopy peak with            wavenumber which is from 2000 cm⁻¹ to 2300 cm⁻¹ or a            predetermined Fourier transform-infrared spectroscopy peak            with wavenumber which is from 2000 cm⁻¹ to 2300 cm⁻¹ ;

    -   (b) removing dNTP analogues not incorporated into the DNA        extension product; and

    -   (c) determining if the dNTP analogue was incorporated into the        primer in step (a) by measuring after step (b) the wavenumber of        the Raman spectroscopy peak or wavenumber of the Fourier        transform-infrared spectroscopy peak of any dNTP analogue        incorporated in step (a), wherein (1) if the dNTP analogue was        incorporated in step (a) determining from the wavenumber of the        Raman spectroscopy peak or wavenumber of the Fourier        transform-infrared spectroscopy peak measured the identity of        the incorporated dNTP analogue and thus determining the identity        of the complementary nucleotide residue in the single-stranded        DNA, and (2) wherein if the dNTP analogue was not incorporated        in step (a) iteratively performing steps (a) through (c) until        the complementary nucleotide residue in the single-stranded DNA        is identified, with the proviso that each dNTP analogue used to        contact the single-stranded DNA template in each subsequent        iteration of step (a), (i) has a predetermined Raman        spectroscopy peak whose wavenumber is different from the        wavenumber of the Raman spectroscopy peak of every dNTP analogue        used in preceding iterations of step (a) or has a predetermined        Fourier transform-infrared spectroscopy peak which is different        from the Fourier transform-infrared spectroscopy peak of every        dNTP analogue used in preceding iterations of step (a), and (ii)        comprises a base which is different from the base of every dNTP        analogue used in preceding iterations of step (a),        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

Still further, this invention concerns a method for determining theidentity of a nucleotide residue within a stretch of consecutive nucleicacid residues in a single-stranded DNA comprising:

-   -   (a) contacting the single-stranded DNA with four different        oligonucleotide probes, (1) wherein each of the oligonucleotide        probes comprises (i) a portion that is complementary to a        portion of consecutive nucleotides of the single stranded DNA        immediately 3′ to the nucleotide residue being identified,        and (ii) a 3′ terminal nucleotide residue analogue comprising on        its sugar a 3′-O—R′ group wherein R′ is (a) is azidomethyl, or a        substituted or unsubstituted hydrocarbyl group and (b) has a        predetermined Raman spectroscopy peak with wavenumber which is        from 2000 cm⁻¹ to 2300 cm⁻¹, and which is different from the        wavenumber of the Raman spectroscopy peak of the R′ of the 3′        terminal nucleotide residue analogue of the other three        oligonucleotide probes, or has a predetermined Fourier-transform        infra red spectroscopy peak with wavenumber which is from 2000        cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of the        Fourier-transform infra red peak of the R′ of the 3′ terminal        nucleotide residue analogue of the other three oligonucleotide        probes, and (iii) each of the four terminal nucleotide residue        analogue comprises a base which is different from the base of        the terminal nucleotide residue analogue of the other three        oligonucleotide probes, and (2) under conditions permitting        hybridization of the primer which is fully complementary to the        portion of consecutive nucleotides of the single stranded DNA        immediately 3′ to the nucleotide residue being identified;    -   (b) removing oligonucleotide primers not hybridized to the        single-stranded DNA; and    -   (c) determining the wavenumber of the Raman spectroscopy peak or        wavenumber of the Fourier-transform infra red peak of the dNTP        analogue of the oligonucleotide probe hybridized in step (a) so        as to thereby determine the identity of the dNTP analogue of the        hybridized oligonucleotide probe and thus determine the identity        of the complementary nucleotide residue in the single-stranded        DNA,        thereby identifying the nucleotide residue within the stretch of        consecutive nucleic acid residues in the single-stranded DNA.

In some embodiments of the invention, the dNTP analogues or R′ groupshave a Raman spectroscopy peak with wavenumber of from 2100 cm⁻¹ to 2260cm⁻¹. In certain embodiments, the wavenumber of the Raman spectroscopypeak is determined by irradiating the incorporated dNTP analogue with532 nm, 633 nm, or 785 nm light. In some embodiments, at least one ofthe primer, probes, or the single-stranded DNA is attached to a solidsurface. In select embodiments, the Raman spectroscopy issurface-enhanced Raman spectroscopy. In some such embodiments, the dNTPanalogue R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom.

In other embodiments of the invention the dNTP analogues each have thestructure:

where R^(x) is a C1-C5 cyanoalkyl, a C1-C5 alkyl, a C2-C5 alkenyl, or aC2-C5 alkynyl, which is substituted or unsubstituted.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in“C1-Cn alkyl” includes groups having 1, 2, . . . , n-1 or n carbons in alinear or branched arrangement. For example, a “C1-C5 alkyl” includesgroups having 1, 2, 3, 4, or 5 carbons in a linear or branchedarrangement, and specifically includes methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, and pentyl.

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon group,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C2-C5 alkenyl” means an alkenyl group having2, 3, 4, or 5, carbon atoms, and up to 1, 2, 3, or 4, carbon-carbondouble bonds respectively. Alkenyl groups include ethenyl, propenyl, andbutenyl.

The term “alkynyl” refers to a hydrocarbon group straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C2-C5alkynyl” means an alkynyl group having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

The term “substituted” refers to a functional group as described abovesuch as an alkyl, or a hydrocarbyl, in which at least one bond to ahydrogen atom contained therein is replaced by a bond to non-hydrogen ornon-carbon atom, provided that normal valencies are maintained and thatthe substitution(s) result(s) in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Non-limiting examples of substituentsinclude the functional groups described above, and for example, N, e.g.so as to form —CN.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

It is understood that where chemical groups are represented herein bystructure, the point of attachment to the main structure is representedby a wavy line.

In the compound structures depicted herein, hydrogen atoms, except onribose and deoxyribose sugars, are generally not shown. However, it isunderstood that sufficient hydrogen atoms exist on the representedcarbon atoms to satisfy the octet rule.

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

A—Adenine;

C—Cytosine;

G—Guanine;

T—Thymine;

U—Uracil;

DNA—Deoxyribonucleic acid;

RNA—Ribonucleic acid;

FTIR—Fourier-transform infrared.

“Nucleic acid” shall mean, unless otherwise specified, any nucleic acidmolecule, including, without limitation, DNA, RNA and hybrids thereof.In an embodiment the nucleic acid bases that form nucleic acid moleculescan be the bases A, C, G, T and U, as well as derivatives thereof.Derivatives of these bases are well known in the art, and areexemplified in PCR Systems, Reagents and Consumables (Perkin ElmerCatalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J.,USA).

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid or an agent may be affixed. Non-limitingexamples include chips, beads, nanopore structures and columns. In anembodiment the solid substrate can be present in a solution, includingan aqueous solution, a gel, or a fluid.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid based on the well-understood principle ofsequence complementarity. In an embodiment the other nucleic acid is asingle-stranded nucleic acid. The propensity for hybridization betweennucleic acids depends on the temperature and ionic strength of theirmilieu, the length of the nucleic acids and the degree ofcomplementarity. The effect of these parameters on hybridization is wellknown in the art (see Sambrook J, Fritsch E F, Maniatis T. 1989.Molecular cloning: a laboratory manual. Cold Spring Harbor LaboratoryPress, New York.). As used herein, hybridization of a primer sequence,or of a DNA extension product, to another nucleic acid shall meanannealing sufficient such that the primer, or DNA extension product,respectively, is extendable by creation of a phosphodiester bond with anavailable nucleotide or nucleotide analogue capable of forming aphosphodiester bond.

As used herein, unless otherwise specified, a base which is “differentfrom” another base or a recited list of bases shall mean that the basehas a different structure from the other base or bases. For example, abase that is “different from” adenine, thymine, and cytosine wouldinclude a base that is guanine or a base that is uracil.

In some embodiments of the invention, vibrational spectroscopy is usedto detect the presence of incorporated nucleotide analogues. Vibrationalspectroscopy is a spectrographic analysis where the sample isilluminated with incident radiation in order to excite molecularvibrations. Vibrational excitation, caused by molecules of the sampleabsorbing, reflecting or scattering a particular discrete amount ofenergy, is detected and can be measured. The two major types ofvibrational spectroscopy are infrared (usually FTIR) and Raman. If FTIRis employed, then the IR spectra of the nucleotide analogues aremeasured (for example of the nucleotide analogues and in the methodsdescribed herein) If Raman is employed, then the Raman spectra of thenucleotide analogues is measured (for example of the nucleotideanalogues and in the methods described herein).

In certain embodiments, the single-stranded DNA, RNA, primer or probe isbound to the solid substrate via 1,3-dipolar azide-alkyne cycloadditionchemistry. In an embodiment the DNA, RNA, primer or probe is bound tothe solid substrate via a polyethylene glycol molecule. In an embodimentthe DNA, RNA, primer or probe is alkyne-labeled. In an embodiment theDNA, RNA, primer or probe is bound to the solid substrate via apolyethylene glycol molecule and the solid substrate isazide-functionalized. In an embodiment the DNA, RNA, primer or probe isimmobilized on the solid substrate via an azido linkage, an alkynyllinkage, or biotin-streptavidin interaction. Immobilization of nucleicacids is described in Immobilization of DNA on Chips II, edited byChristine Wittmann (2005), Springer Verlag, Berlin, which is herebyincorporated by reference. In an embodiment the DNA is single-strandedDNA. In an embodiment the RNA is single-stranded RNA.

In other embodiments, the solid substrate is in the form of a chip, abead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, aporous media, a porous nanotube, or a column. This invention alsoprovides the instant method, wherein the solid substrate is a metal,gold, silver, quartz, silica, a plastic, polypropylene, a glass, ordiamond. This invention also provides the instant method, wherein thesolid substrate is a porous non-metal substance to which is attached orimpregnated a metal or combination of metals. The solid surface may bein different forms including the non-limiting examples of a chip, abead, a tube, a matrix, a nanotube. The solid surface may be made frommaterials common for DNA microarrays, including the non-limitingexamples of glass or nylon. The solid surface, for examplebeads/micro-beads, may be in turn immobilized to another solid surfacesuch as a chip.

In one embodiment, the solid surface is a SERS-prepared surface designedspecifically for detection of a label nucleotide. The surface mayinclude one or more nanoplasmonic antenna, wherein the nanoplasmonicantenna may be a nanoplasmonic bowtie antenna. In one embodiment, thenanoplasmonic bowtie antenna comprises crossed-bowtie structure in whichone pair of triangles couples to incident field, while another pair oftriangles couples to Raman scattered field in an orthogonalpolarization. It is also contemplated that the nanoplasmonic antenna maybe an array of antennas. In addition, the nanoplasmonic antenna mayinclude DNA functionalized sites, and may have a gap size range from 50nm to 8 nm. In another embodiment, a DNA polymerase is immobilizedwithin the gap.

In another embodiment, the surface comprises a DNA origami scaffold oran array of DNA origami scaffolds. It is also contemplated that the DNAorigami scaffold further comprising a primer molecules positionedbetween Au and Ag nanoparticles and nanorods located at specifiedbinding sites.

In a further embodiment, the surface comprises plasmonic crystals or anarray of plasmonic structures. For example, the plasmonic structures maybe periodic TiO-Au-TiO structures.

Also disclosed herein is a process for producing the solid surface ofany one of claims 50-63, which process comprising:

-   -   a) performing a first lithography step to etch into substrate;    -   b) depositing Ag or Al with a Ti cap by electron beam        evaporation and lifting off the substrate;    -   c) passivating the solid surface with a CVD oxidation layer;    -   d) depositing hexamethyl-disilazane (HMDS);    -   e) performing a second lithography step to define a primer        adhesion site; and    -   f) removing HMDS,        so as to produce the surface enhanced Raman scattering (SERS)        solid surface.

In various embodiments the nucleic acid samples, DNA, RNA, primer orprobe are separated in discrete compartments, wells or depressions on asurface.

In this invention methods are provided wherein about 1000 or fewercopies of the nucleic acid sample, DNA, RNA, primer or probe, are boundto the solid substrate. This invention also provides the instantinvention wherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of thenucleic acid sample, DNA, RNA, primer or probe are bound to the solidsubstrate.

In some embodiments, the immobilized nucleic acid sample, DNA, RNA,primer or probe is immobilized at a high density. This invention alsoprovides the instant invention wherein over or up to 1×10⁷, 1×10⁸, 1×10⁹copies of the nucleic acid sample, DNA, RNA, primer or probe, are boundto the solid substrate.

In other embodiments of the methods and/or compositions of thisinvention, the DNA is single-stranded. In an embodiment of the methodsor of the compositions described herein, the RNA is single-stranded.

In certain embodiments, UV light is used to photochemically cleave thephotochemically cleavable linkers and moieties. In an embodiment, thephotocleavable linker is a 2-nitrobenzyl moiety. In an embodiment of theprocesses and methods described herein monochromatic light is used toirradiate Raman-label-containing nucleotide analogues (e.g. incorporatedinto a primer or DNA extension product) so as to elicit a signalmeasurable by Raman spectroscopy. In one such embodiment, the laser is a532 nm, 633 nm, or 785 nm laser. In another such embodiment, nearinfra-red light is used to irradiate Raman-label-containing nucleotideanalogues. In certain embodiments of the processes and methods of thisinvention near infra-red light is used to irradiateRaman-label-containing polynucleotide analogues.

Methods for production of cleavably capped and/or cleavably linkednucleotide analogues are disclosed in U.S. Pat. No. 6,664,079, which ishereby incorporated by reference.

A “nucleotide residue” is a single nucleotide in the state it existsafter being incorporated into, and thereby becoming a monomer of, apolynucleotide. Thus, a nucleotide residue is a nucleotide monomer of apolynucleotide, e.g. DNA, which is bound to an adjacent nucleotidemonomer of the polynucleotide through a phosphodiester bond at the 3′position of its sugar and is bound to a second adjacent nucleotidemonomer through its phosphate group, with the exceptions that (i) a 3′terminal nucleotide residue is only bound to one adjacent nucleotidemonomer of the polynucleotide by a phosphodiester bond from itsphosphate group, and (ii) a 5′ terminal nucleotide residue is only boundto one adjacent nucleotide monomer of the polynucleotide by aphosphodiester bond from the 3′ position of its sugar.

Because of well-understood base-pairing rules, determining thewavenumber of the Raman spectroscopy peak or wavenumber of the FTIR peakof a dNTP analogue incorporated into a primer or DNA extension product,and thereby the identity of the dNTP analogue that was incorporated,permits identification of the complementary nucleotide residue in thesingle stranded polynucleotide that the primer or DNA extension productis hybridized to. Thus, if the dNTP analogue that was incorporated has aunique wavenumber in the Raman spectroscopy peak identifying it ascomprising an adenine, a thymine, a cytosine, or a guanine, then thecomplementary nucleotide residue in the single stranded DNA isidentified as a thymine, an adenine, a guanine or a cytosine,respectively. The purine adenine (A) pairs with the pyrimidine thymine(T). The pyrimidine cytosine (C) pairs with the purine guanine (G).Similarly, with regard to RNA, if the dNTP analogue that wasincorporated comprises an adenine, a uracil, a cytosine, or a guanine,then the complementary nucleotide residue in the single stranded RNA isidentified as a uracil, an adenine, a guanine or a cytosine,respectively.

Incorporation into an oligonucleotide or polynucleotide (such as aprimer or DNA extension strand) of a dNTP analogue means the formationof a phosphodiester bond between the 3′ carbon atom of the 3′ terminalnucleotide residue of the polynucleotide and the 5′ carbon atom of thedNTP analogue resulting in the loss of pyrophosphate from the dNTPanalogue.

As used herein, a deoxyribonucleoside triphosphate (dNTP) analogue,unless otherwise indicated, is a dNTP having substituted in the 3′-OHgroup of the sugar thereof, in place of the H atom of the 3′-OH group,or connected via a linker to the base thereof, a chemical group whichhas Raman spectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300cm⁻¹ or FTIR peak wavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹and whichdoes not prevent the dNTP analogue from being incorporated into apolynucleotide, such as DNA, by formation of a phosphodiester bond.Similarly, a deoxyribonucleotide analogue residue is deoxyribonucleotideanalogue which has been incorporated into a polynucleotide and whichstill comprises its chemical group having a Raman spectroscopy peak withwavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹ or FTIR peak with wavenumberof from 2000 cm⁻¹ to 2300 cm⁻¹. In a preferred embodiment of thedeoxyribonucleoside triphosphate analogue, the chemical group issubstituted in the 3′-OH group of the sugar thereof, in place of the Hatom of the 3′-OH group. In a preferred embodiment of thedeoxyribonucleotide analogue residue, the chemical group is substitutedin the 3′-OH group of the sugar thereof, in place of the H atom of the3′-OH group. In an embodiment the chemical group has a Ramanspectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹.

As used herein, a ribonucleoside triphosphate (rNTP) analogue, unlessotherwise indicated, is an rNTP having substituted in the 3′-OH group ofthe sugar thereof, in place of the H atom of the 3′-OH group, orconnected via a linker to the base thereof, a chemical group which has aRaman spectroscopy peak with wavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹or FTIR peak with wavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹ and whichdoes not prevent the rNTP analogue from being incorporated into apolynucleotide, such as RNA, by formation of a phosphodiester bond.Similarly, a ribonucleotide analogue residue is ribonucleotide analoguewhich has been incorporated into a polynucleotide and which stillcomprises its chemical group having a Raman spectroscopy peak withwavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹ or FTIR peak with wavenumberof from 2000 cm⁻¹ to 2300 cm⁻¹. In a preferred embodiment of theribonucleoside triphosphate analogue, the chemical group is substitutedin the 3′-OH group of the sugar thereof, in place of the H atom of the3′-OH group. In a preferred embodiment of the ribonucleotide analogueresidue, the chemical group is substituted in the 3′-OH group of thesugar thereof, in place of the H atom of the 3′-OH group. In anembodiment the chemical group has a Raman spectroscopy peak withwavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹.

A Raman spectroscopy system, as can be used in the methods describedherein, typically comprises an excitation source (such as a laser,including a laser diode in appropriate configuration, or two or morelasers), a sample illumination system and light collection optics, awavelength selector (such as a filter or spectrophotometer), and adetection apparatus (such as a CCD, a photodiode array, or aphotomultiplier). Interference (notch) filters with cut-off spectralrange of ±80-120 cm-1 from the laser line can be used for stray lightelimination. Holographic gratings can be used. Double and triplespectrometers allow taking Raman spectra without use of notch filters.Photodiode Arrays (PDA) or a Charge-Coupled Devices (CCD) can be used todetect Raman scattered light. In an embodiment, surface enhanced Ramanspectroscopy (SERS) is used which employs a surface treated with one ormore of certain metals known in the art to cause SERS effects. In anembodiment the surface is a surface to which the polynucleotide,single-stranded DNA, single-stranded RNA, primer, DNA extension strandor oligonucleotide probe of the methods described herein is attached.Many suitable metals are known in the art. In an embodiment the surfaceis electrochemically etched silver or treated with/comprises silverand/or gold colloids with average particle size below 20 nm. Thewavenumber of the Raman spectroscopy peak of an entity is identified byirradiating the entity with the excitation source, such as a laser, andcollecting the resulting Raman spectrum using a detection apparatus. Thewavenumber of the Raman spectroscopy peak is determined from the Ramanspectrum. In an embodiment, the spectrum measured is from 2000 cm⁻¹ to2300 cm⁻¹ and the wavenumber of the Raman spectroscopy peak is the peakwavenumber within that spectrum. In an embodiment the spectrum measuredis a sub-range of 2000 cm⁻¹ to 2300 cm⁻¹ and the Raman spectroscopy peakwavenumber is the peak wavenumber within that spectrum sub-range.

FTIR systems as can be used with the FTIR methods described herein arewell-known in the art, for example grazing angle FTIR.

Where a range of values is provided, unless the context clearly dictatesotherwise, it is understood that each intervening integer of the value,and each tenth of each intervening integer of the value, unless thecontext clearly dictates otherwise, between the upper and lower limit ofthat range, and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding (i) either or (ii)both of those included limits are also included in the invention.

All combinations of the various elements described herein are within thescope of the invention. All sub-combinations of the various elementsdescribed herein are also within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

Here an enhanced high-sensitivity, high-resolution detection system,Surface-Enhanced Raman Scattering (SERS), which takes advantage ofunique 3′-O-azidomethyl-modified nucleotide reversible terminators(3′-O—N₃-NRTs), is used for SBS. The SERS detection sensitivity canapproach single molecule level.¹⁷ Thus SERS-SBS is able to provide ahigh-throughput and high-sensitivity SBS approach, which complementsfluorescent SBS and pyrosequencing. The methods and compositionsdescribed herein below can be alternatively be used for FTIR-baseddetection, mutatis mutandis. As seen in the results herein, a library oftags have been identified with Raman peaks in the region of200-2300cm⁻¹, preferably 2100-2250 cm⁻¹, where DNA and proteins have noRaman peaks. It is also demonstrated that using the azidomethyl group isexcellent in terms of sensitivity.

Because these 3′-O—N₃-NRTs do not require the attachment of fluorescenttags, their cost of synthesis is substantially less than those that do.They are much smaller than their counterparts with fluorescent tags,which increase their incorporation efficiency by DNA polymerase. Aftercleavage, the N₃ is completely destroyed yielding no Raman signal at˜2100 cm⁻¹. In addition, the extended chain is identical to natural DNA.Many current approaches for SBS require the use of modified nucleotidesthat leave short remnants of the linkers that were used to attach thefluorescent tags; as these build up in the extended DNA chains, they aremore and more likely to alter the DNA structure so as to impede furthernucleotide incorporation. Finally, the conditions for removal of theazidomethyl group to allow the next cycle of incorporation and detectionis well established, and has been shown to be compatible with DNAstability (15, 18).

SERS-SBS is a unique approach that to our knowledge has not been testedfor DNA sequencing outside of our laboratory. There are severalinnovative aspects all accomplished by the use of 3′-O—N₃-NRTs in thissequencing strategy: (1) a ready mode of detection available in theSurface-Enhanced Raman Scattering technique; (2) a means of overcomingerrors, particularly in reading through homopolymer stretches, thanks tothe presence of the reversible terminating group; (3) the elimination ofthe problem of fluorescence background; (4) increased processivity ofthe enzyme reaction thanks to the absence of any modifications on thepreviously incorporated nucleotides; (5) relatively low cost ofsynthesis compared to fluorescently tagged nucleotides; and (6) ease ofremoval of the azidomethyl moiety with established and DNA-compatiblechemistry (TCEP cleavage of the azidomethyl group restores the —OH).Further innovation in the use of the SERS technique is achieved by itshigh sensitivity and excellent resolution.

A further innovation is the use of a library of different nucleotideswith distinct chemical side groups on the azidomethyl moiety, one foreach of the 4 bases. These are chosen so as to generate unique ormultiple Raman band shifts and therefore unique signatures for eachbase, analogous to 4-color DNA sequencing. Identification of whichanalogues can perform these functions and be incorporated by apolymerase is critical.

Thus, a major innovation here is a technology that includes both costand throughput advantages with the goal of achieving the $1000 genome,but also doing this at high sensitivity, and increasing the number ofDNA molecules that can be sequenced in parallel. It is apparent that thesame molecules can be utilized as well for non-genomic sequencing, suchas direct RNA-Seq. In fact, the use of the SERS approach, with theazidomethyl moiety located instead at positions that do not causetermination, is compatible with other approaches, includingexonuclease-based sequencing.

DNA Sequencing by Synthesis using Reversible Terminators with CleavableFluorescent Tags:

Over the last 10 years, a wide variety of chemistries and technologiesto support the sequencing by synthesis (SBS) strategy have beendeveloped. This includes the use of fluorescent and mass tags to revealthe specific incorporation of nucleotides containing each of the 4bases, various surface attachment strategies to enable solid-phase SBS,and sophisticated hybrid strategies (mixed dNTP/NRT strategies tomaximize length, pyrosequencing with NRTs, a novel walking andsequencing strategy using the SBS platform) (10, 14-16, 22-26). Sincemuch of this work has been published, here only the basic approach isbriefly described with some specific examples.

Upon examining X-ray diffraction-based models of the interactions of aDNA template, DNA primer, and an incoming dNTP at the reaction center ofDNA polymerase (28), it became apparent that only a few sites on thenucleotide were sufficiently free of steric hindrance and ionicinterference to support the attachment of side groups and still permitthe polymerase reaction to occur with good efficiency and specificity.By attaching small cleavable chemical groups to the 3′-OH site on thesugar, and larger fluorescent or mass tags to the bases via cleavablelinkers, libraries of molecules were created that could temporarily stopthe incorporation of additional nucleotides, and provide a specificmeans of identifying the incorporated nucleotide. Subsequently, both theblocking group and the tags could be removed by chemical orphotocleavage reactions, in preparation for the next round of DNAincorporation (10,14,18). Among the variety of 3′-OH modificationstested, three were followed with particular avidity, based on theirefficiency of incorporation, ease of cleavage with agents that were notdamaging to the DNA, and specificity. These included the 2-nitrobenzylgroup which could be removed by exposure to near-UV light (23) the allylgroup which could be cleaved via a Pd-catalyzed reaction (14) and theazidomethyl group which is cleavable by treatment under mild conditionswith Tris-(2-carboxyethyl)phosphine (TCEP) (15). For attachment of thefluorescent labels, an assortment of linkers that also incorporatedthese three groups allowing cleavage by the same means were generated.

In FIG. 1, the approach with a set of 4 nucleotides possessing bothcleavable tags and cleavable blocking groups is shown, using forsimplicity the class of compounds containing an azido group at the 3′-OHand within the linker to the fluorophores (18). As shown, after aninitial polymerase reaction, the hybridized primer is extended by anucleotide complementary to the next available base in the template DNA.Because of the presence of the blocking group, the reaction isterminated at that position, and because of its specific fluorescenttag, the base can be determined. Subsequently, TCEP is added to cleaveboth the blocking group and the fluorescent tag, at which point thestage is set for the next round of sequencing(incorporation-detection-cleavage). Both short and longer flexiblelinkers have been utilized to attach the fluors, which upon cleavageleave a smaller remnant. This is important as these remnants may have acumulative effect on the structure of the growing DNA chain that willeventually make it difficult for the polymerase to recognize it as agood substrate for further reactions, thereby placing a ceiling on thepotential read length. Also shown in FIG. 1 is the use of anintermediate synchronization step in which NRTs without attached dyesare added; these are more efficiently incorporated than the labeledNRTs, and allow incomplete (“lagging”) reactions from the prior step to“catch up”.

In another approach, a hybrid between the massively parallelpossibilities of SBS and the long sequence reads enabled by Sangersequencing, 3′-O-modified nucleotide reversible terminators and muchsmaller amounts of chemically cleavable fluorescent dideoxynucleosides(ddNTPs) which permanently terminate the reaction have been utilized atthe same time (15). The principle is that sufficient signal is obtainedfrom the labeled permanent terminators, even though the small percent ofstrands into which they are incorporated are lost from future reactions.In the meantime, the reversible terminators are available to drivesubsequent steps, and because the free 3′-OH groups are regenerated ineach round, and there are no remnant-leaving linkers on these NRTs, thereactions should progress smoothly. By varying the ratios of the labeledddNTPs and the unlabeled NTPs in each round, longer sequencing readsthan with NRTs with attached dyes have been achieved.

DNA Sequencing by Synthesis using 3′-azidomethyl Modified NRTs andInfrared Spectroscopic (IR) Detection.

Here the unique infrared absorbance at 2115 cm⁻¹ of the azido group (N₃)in the 3′-azidomethyl modified NRTs (FIG. 2) is disclosed for DNAsequence detection. In this approach, the incorporation of the3′-azidomethyl modified NRTs into the growing strand of DNA temporarilyterminates the polymerase reaction. Instead of engineering a fluorescentdye as a reporter group on the bases, or detecting the releasedpyrophosphate, here the azidomethyl capping moiety on the 3′-OH servesdouble duty: as the reversible termination group (FIG. 3), and also asthe reporter group to indicate the incorporation of the complementarybase. The infrared spectrum for the azido (N₃) group (˜2115 cm⁻¹) isstrong and unique while none of the groups in DNA have IR signals inthis region. Thus, N₃ serves as an IR label for DNA sequencing bysynthesis (SBS) (FIG. 4). Compared with pyrosequencing, IR-SBS using3′-azidomethyl modified NRTs has the following advantages: (1)Throughput is much higher than pyrosequencing, because the N₃ tag isdirectly attached on the DNA, which allows a higher density of DNAtemplates to be attached on a solid surface without any cross talk; (2)All the growing strands in IR-SBS are natural DNA molecules and sequencedetermination is a direct detection approach; in contrast,pyrosequencing is an indirect detection method; (3) the sequencingdevice can be miniaturized as it only requires a narrow spectral regionaround 2100 cm⁻¹.

Because the N₃ group in the 3′-azidomethyl modified NRTs also has aRaman band at ˜2100 cm⁻¹, Surface-enhanced Raman scattering (SERS) ofthe N₃ group can be used for an SERS-SBS approach. The SERS detectionsensitivity can approach single molecule level (17). Thus SERS-SBS isable to provide a high-throughput and high-sensitivity SBS approach,which complements fluorescent SBS and pyrosequencing.

DNA Sequencing by Synthesis using 3′-azidomethyl Modified NRTs and RamanDetection.

As an initial test of the potential of the Raman detection with SBS,four model compounds were selected with 4 different Raman tags (—N₃,—CN, —C≡CH, and —C≡C—CH₃) as shown in FIG. 5. As seen in the spectra,appropriate fairly sharp Raman peaks with each of these compounds wereobtained. The N₃ peak appears at ˜2105 cm⁻¹, the alkyne peak at ˜2138cm⁻¹, the methyl-substituted alkyne peak at ˜2249 cm⁻¹, and the cyanopeak at ˜2259 cm⁻¹. Importantly, in compounds with two different tagsand in compound mixtures, all the expected peaks appear and at nearequal stoichiometry at the appropriate wavenumber, where DNA and proteinhave no Raman peaks.

Surfaced Enhanced Raman Scattering (SERS) of Model Compounds.

SERS has been shown to be able to detect a variety of molecules at thesingle molecule level. For example, recent reports provide evidence thatsingle molecular detection (SMD) is strongly linked to localized surfaceplasmonic resonance (LSPR) supporting nanostructures; single moleculeRaman detection of a cyano group (—C≡N) in Rhodamine 800 (FIG. 6) (33);and single-molecule surface-enhanced Raman spectroscopy of crystalviolet obtaining an overall enhancement of 2.6×10⁹ from eight SM-SERSevents (34). In addition, it has been shown that <100 molecules weredetectable using urchin-like silver nanowire as SERS substrate and 10⁻¹⁶M surface adsorbed Rhodamine 6G (30).

Raman and SERS Experiments.

Test compounds synthesized (2-azido-3-(benzyloxy)propanoic acid (C1)containing an azido (—N₃) group and 2-cyanoethyl2-azido-3-(benzyloxy)propanoate (C2) containing both an N₃ and cyano(—C≡N) moiety (FIG. 5)) were dissolved in methanol and diluted. Analiquot was applied to a SERS substrate or glass slide for measurement.An aluminum reference surface was used as a control. The Klarite(Renishaw Diagnostics, Ltd.) substrate consisting of a 4 mm×4 mmnano-patterned SERS active area in which Au was layered in an invertedpyramid array (FIG. 7) was used as the SERS surface. Typically, fivemeasurements were obtained across each substrate surface.

Characterization of SERS Enhancement Factors.

Raman signals of the C1 and C2 adsorbed samples were measured andcompared with the aluminum reference substrate. The analyticalenhancement factor (AEF) was defined as:

${A\; E\; F} = \frac{I_{SERS}/c_{SERS}}{I_{RS}/c_{RS}}$

where:

-   -   I_(SERS)=SERS signal intensity    -   I_(RS)=Raman signal intensity of aluminum reference    -   c_(SERS)=analyte solution concentration under non-SERS condition    -   c_(RS)=analyte concentration on SERS substrate

Results of Model Raman Tag Measurements.

Using various analyte dilutions, an area-average SERS enhancement of˜8×10⁵ (N₃ compound) and ˜5×10⁵ (CN compound) were achieved over theentire SERS-active area. The averaged and background removed Raman peaksfor the azido compound is shown in FIG. 8 for both Klarite (red) andaluminum reference (blue). The resulted EF was close to the definedtheoretical maximum of ˜10⁶. The results may be further improved using ananoengineering approach to create optimal SERS surfaces or plasmonicsystems.

Successful DNA SBS using SERS with a commercially available structuredgold surface and a synthetic template and primer has been shown. In thisSERS-SBS approach, the 3′-OH groups of the reporter nucleotides arecapped with an azidomethyl moiety (15), as shown in FIG. 24, totemporarily terminate the polymerase reaction after incorporation.Instead of engineering a fluorescent dye as a reporter group on thebases, here the same azidomethyl capping moiety serves as the reportergroup to indicate the incorporation of a base complementary to that inthe template into a growing DNA chain. The azide group is ideally suitedas a Raman reporter group, because of its distinct Raman shift at2080-2170 cm⁻¹, a spectral region where DNA, proteins and most othermolecules do not elicit signals.

Because these 3′-O-azidomethyl-dNTPs (3′-O—N₃-dNTPs) do not require theattachment of fluorescent tags, their cost of synthesis is substantiallylower. They are much smaller than their counterparts with fluorescenttags, which increase their incorporation efficiency by DNA polymerase.In addition, the extended chain is identical to natural DNA, unlike manycurrent SBS approaches, which require the use of modified nucleotidesthat leave short remnants of the linkers used to attach the fluorescenttags; as these build up in the extended DNA chains, they areincreasingly likely to alter DNA structure and impede further nucleotideincorporation. After cleavage, the N₃ is completely destroyed yieldingno Raman signal at ˜2125 cm⁻¹. Finally, the method for removal of theazidomethyl group to allow the next cycle of incorporation and detectionis well established and has been shown to be highly compatible with DNAstability (15).

Before proceeding with the SBS experiment, the four3′-O-azidomethyl-modified nucleotides are shown to generate appropriateRaman signals relative to the natural nucleotides. These serve to mimicthe nucleotides incorporated into the growing strand of DNA before andafter TCEP cleavage, respectively. Indeed, the four 3′-O—N₃-dNTPsdisplay enhanced Raman scattering at ˜2125 cm⁻¹ on Klarite SERSsubstrates, while the natural dNTPs produce only a background signal(FIG. 9).

Consecutive incorporation, detection and cleavage of each of the fournucleotides bearing 3′-O-azidomethyl blocking groups (N₃) using atemplate and linear primer is successful. The approach took advantage ofa template-primer combination in which the next four nucleotides to beadded were A, C, G and T. As shown in FIG. 10 (middle), a3′-O-azidomethyl modified complementary base could be incorporated intoa 13-mer primer annealing to a 51-mer DNA template. After removing allthe reaction components by HPLC, the appearance of Raman spectra around2125 cm⁻¹ indicated the incorporation of 3′-O—N₃-dNTPs into the DNAstrand. Treatment with a 100 mM TCEP (tris(2-carboxyethyl)phosphine)solution was able to remove the azidomethyl group and regenerate the3′-OH of the DNA primer, after which incorporation could be initiatedfor the next cycle. The experiment was initiated with the 13-mer primerannealed to a DNA template. When the first complementary base,3′-O—N₃-dATP, was used in the polymerase reaction (FIG. 10, middle), theRaman spectra of the extended DNA template clearly showed a Raman shiftat ˜2125 cm⁻¹ which can be assigned to the azide stretch (FIG. 10 a,left); the expected 4329 Da extension product was confirmed by MALDI-TOFMS (FIG. 10 a, right). This is strong evidence that the modifiednucleotide was incorporated into the DNA primer. After TCEP treatment toremove the azidomethyl group and HPLC purification, the Raman peakaround 2125 cm⁻¹ largely disappeared, and cleavage was confirmed by anMS peak at 4274 Da (FIG. 10 b). The newly formed free 3′-OH containingprimers were then used in a second polymerase cycle where 3′-O—N₃-dCTPwas added. The Raman spectra again revealed a peak at ˜2125 cm⁻¹, and MSgave a 4621 Da peak, indicating incorporation of 3′-O—N₃-dCTP in thiscycle (FIG. 10 c); disappearance of the Raman peak after TCEP treatmentand an MS peak at 4566 Da proved the removal of the azidomethyl group(FIG. 10 d). FIG. 10 e showed the third incorporation of 3′-O—N₃-dGTPinto this primer to resume the N₃-dependent Raman signal and thedisappearance of this signal after TCEP cleavage is indicated in FIG. 10f. Finally, 3′-O—N₃-dTTP was incorporated in the polymerase reaction inthe fourth cycle, as shown in FIG. 10 g, indicated by the reappearanceof the N₃ Raman signal, and FIG. 10 h shows the disappearance of thispeak after azidomethyl group removal by TCEP. Again, appropriate masseswere obtained by MALDI-TOF-MS for the 3^(rd) and 4^(th) incorporationand cleavage reactions.

DNA sequencing by synthesis using surface-enhanced Raman spectroscopyhas been shown. The azidomethyl group at the 3′-OH position of thenucleotides can temporarily terminate the polymerase reaction after theyare incorporated and treatment with TCEP can efficiently remove theazidomethyl group from the 3′-OH. Meanwhile, the uniqueness of the azidepeak in Raman spectra makes it an excellent reporter group during SBS.Once the complementary base was incorporated into the DNA strand, aRaman peak appeared around 2125 cm⁻¹, otherwise, this portion of thespectrum was at background level. This method of DNA sequencing isrealized with only minor nucleotide modifications. The nucleobases areintact and the DNA primer remains in its natural form as it's elongatedduring SBS, therefore, a longer readlength would be achieved. Thougheach reaction was carried out in solution and then purified and spottedon separate Klarite chips, SBS experiments can also follow continuousincorporation and cleavage on the same chip. Additionally, integrationof nanoplasmonic systems together with site specific molecularinteractions to yield reproducible and optimal signal enhancement can beachieved by attaching either the DNA or the polymerase to the SERSsurface, permitting washing away of unreacted nucleotides and otherreactants.

The high spectral quality and reproducibility of the DNA extensionproduct spectra, which are clearly distinguishable from the Ramanspectra of the cleavage products, may be used to develop a robustsolid-state platform for measuring SERS signals resulting from thepolymerase extension of specific oligonucleotides in SBS reactions.Furthermore, with different Raman scattering groups bearing uniquespectral signatures on each of the four nucleotides, one can overcomethe need to add them one at a time.

Experimental Procedures

SERS Detection of Nucleotides

The four 3′-O-azidomethyl-modified nucleotides (FIG. 24) weresynthesized according to the procedure described previously (4). Eachwas dissolved in water to form a 100 μM solution, then a 2 μL aliquotwas deposited onto individual Klarite SERS (Renishaw Diagnostics, UK)substrates and dried in ambient air to obtain a uniform moleculardeposition for Raman measurements. Equivalent amounts of the naturaldeoxynucleotides were deposited on 4 separate SERS substrates in thesame way.

Sequencing by Synthesis (SBS) Reactions

Polymerase extension reactions each consisted of 20 pmol of a synthetic51-mer DNA template(5′-GAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACC-3′), 60 pmol ofprimer (5′-CACATTGTCAAGG-3′) or a previously extended and TCEP-cleavedproduct, 100 pmol of a single nucleotide reversible terminator(3′-O—N₃-dATP, 3′-O—N₃-dCTP, 3′-O—N₃-dGTP, or 3′-O—N₃-dTTP) (4), 1×ThermoPol reaction buffer (New England Biolabs, Mass.), 2 unitTherminator™ III DNA polymerase and deionized H₂O in a total volume of20 μL. Reactions were conducted in a thermal cycler (MJ Research, MA).After initial incubation at 94° C. for 20 sec, the reaction wasperformed for 36 cycles at 80° C. for 20 sec, 45° C. for 40 sec and 65°C. for 90 sec.

After the reaction, a small portion of the DNA extension product wasdesalted using a C18 ZipTip column (Millipore, Mass.) and analyzed byMALDI-TOF MS (ABI Voyager, DE). The remaining product was concentratedfurther under vacuum and purified by reverse phase HPLC on an XTerra MSC18 2.5 μm 4.6 mm×50 mm column (Waters, Mass.) to obtain the pureextension product (retention time ˜29 min). Mobile phase: A, 8.6 mMtriethylamine/100 mM 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) in water(pH 8.1); B, methanol. Elution was performed at 40° C. with a 0.5 mL/minflow rate, and with 88% A/12% B to 65.5% A/34.5% B linear gradient for90 min, then 100% B isocratic for another 20 min. The purified productwas used in the subsequent extension reaction.

Cleavage reactions were carried out by dissolving 100 pmol extensionproducts in 10 μL of 100 mM Tris(2-carboxyethyl)phosphine (TCEP)solution (pH 9.0), and incubating at 65° C. for 25 min to remove theazidomethyl groups. Following dilution in 1 mL deionized H₂O anddesalting in an Amicon Ultra-0.5 centrifugal filter unit with Ultracef-3membrane (Millipore), 2 μL was used to obtain the MALDI-TOF massspectrum. Each cleavage product was used as primer in the subsequentextension reaction. The mechanism of this cleavage reaction is shown inFIG. 25. The third and the fourth extensions were carried out in asimilar manner by using the previously extended and cleaved product asthe primer. Four consecutive nucleotide additions are shown in FIG. 26.

SERS Detection of SBS Products

Raman spectra of the newly purified DNA extension and cleavage productswere acquired with a drop coating method, in which an aliquot wasdeposited and dried in ambient air to obtain a uniform layer. A 3 μLaliquot of the 100 μM DNA extension products was deposited onto aKlarite SERS-active substrate (Renishaw Diagnostics); similarly, 30 pmolof cleavage products with 10 pmol spiked-in template were spotted. Theadded DNA was included for compatibility with the extension products,and to more closely mimic a continuous extension and cleavage reactioncarried out on the same substrate.

Description of SERS Substrates

Gold-coated Klarite SERS-active substrates were purchased from RenishawDiagnostics. The 6 mm×10 mm chip (consisting of a 4 mm×4 mm patternedregion and an unpatterned Au reference area) was adhered to a standardmicroscope slide (25 mm×75 mm) at the foundry. The active area containsan array of micro-scaled inverted pyramids with 1.5 μm well diameter, 2μm pitch and 1 μm depth coated with a 20 nm chrome adhesion layer belowa 400 μm gold layer. Klarite slides were used only once and the storagecontainer was opened just prior to measurement to reduce possiblesurface contamination.

Instrumentation

All Raman/SERS spectra were recorded using a Jobin-Yvon LabRam ARAMISRaman microscope (Horiba, Japan) in a standard backscatteringconfiguration with a 785 nm excitation laser. The laser beam was focusedonto the sample using a 50× long working-distance (NA=0.5) dry objective(Nikon, Japan). All spectra were obtained with an exposure time of 10sec, 5 accumulations per spot and at 34 mW laser power before theobjective.

Analysis of Spectral Data

Due to potential non-uniformity of analyte deposition, a data set of Nspectra (in this case, typically N=10) acquired at randomly selectedregions on the same substrate was obtained. Data are presented asbackground removed averages of such a data set. Spectra were processedusing the Savitzky-Golay fourth derivative method (window size of 25data points), which can effectively reduce or eliminate possible falsecorrelations resulting from a constant offset or broadband background(36, 37).

Experiments

Nucleotide reversible terminators (NRTs), 3′-O-azidomethyl derivativesof each of the four nucleotides, and a new set of compounds based on the3′-O-azidomethyl nucleotides but further modified to produce additionalRaman signals were designed. The Raman spectra of the set ofazidomethyl-derivatized NRTs are characterized, as free nucleotides andwhen part of DNA chains, before and after cleavage of the N₃ group. NewSERS substrates specially designed for detecting the Ramantag-containing nucleotides used in these studies are developed. SBS isconducted on surface-immobilized DNA templates using the 3′-N₃-NRTs anddetection by Raman. A set of 4 NRTs possessing 4 unique Raman signaturesare tested, one for each of the four bases of DNA; and analyzed as freenucleotides and when incorporated into DNA, before and after cleavage ofthe Raman detectable groups. Surface-enhanced signals are utilized, forinstance the use of silver or gold nanoparticle-impregnated porousalumina surfaces (nanotubes) (29-31) attached to the sequencing chips,in order to achieve high-sensitivity detection. The system forsequencing of both surface-attached synthetic and library-derived DNAmolecules will be tested.

Preparation of a Set of Nucleotides containing 3′-O-azidomethylModifications and Testing Them for SBS in Solution with Raman and SERSDetection

A set of 3′-O-azidomethyl derivatives of each of the four nucleotideshas previously been synthesized (15). These are recognized as substratesfor SBS by 9° N DNA polymerase (exo-) A485L/Y409V (New England Biolabs)with modified buffer conditions. It has been shown that these NRTs canbe used to conduct SBS reactions without the use of contextinformation-based background subtraction (15). These NRTs have been usedsuccessfully for carrying out primer walking reactions during SBS,offering the potential to substantially increase the current sequencingread length of SBS. In the past these NRTs have either been used incombination with fluorescent nucleotides, or have been further modifiedwith cleavable linkers containing fluorescent tags. Though fluorescencemeasurements are highly sensitive, with the possibility of singlemolecule detection, they are plagued by background issues. Anyfluorescent labels that have not been cleaved and efficiently washedaway from the flow cells in which reactions and fluorescent scanningoccur, can interfere with the ability to obtain clear readings insubsequent sequencing cycles.

Recognizing this limitation of fluorescence measurements andappreciating that the N₃ group produces a strong Raman signal at ˜2100cm⁻¹ where DNA and protein have no Raman peaks, use of thenon-fluorescently labeled 3′-O-azidomethyl nucleotides for DNAsequencing with Raman detection is tested. The azido linkage is easilyand completely cleaved by incubation with Tris-(2-carboxyethyl)phosphine(TCEP) under aqueous conditions (FIG. 3), leaving DNA intact. Inaddition, the cleavage reaction completely destroys the N₃ group,leading to no background Raman signal. After this cleavage, the 3′-OHgroup is restored, and there is no chemical remnant of the blockinggroup; this is important as accumulation of even small modifications oneach nucleotide can affect the curvature and other structural propertiesof the growing DNA chain, potentially interfering with subsequentnucleotide incorporation, leading to earlier termination and shortersequence reads than would otherwise be obtained.

(a) Generation of 3′-O-azidomethyl-dNTPs:

The protocol for synthesis of these NRTs has previously been describedin detail.¹⁵ In brief, the 5′-position and active amino groups on thenucleoside bases are protected, and the 3′-OH group ismethylthiomethylated, reacted with sulfuryl chloride, and then withsodium azide to generate the 3′-O-azidomethyl group.

(b) Solution Raman Scattering Measurement:

Initially, the 3′-O-azidomethyl nucleotides (3′-N₃-NRTs) are tested,characterized by MS and NMR for quality assurance, in solution.Sufficient concentrations are used to obtain detailed spectra withazido-dependent Raman shifts in the expected range (˜2100 cm⁻¹) for eachof the four nucleotides. For comparison, standard dNTPs or ddNTPs,lacking this Raman label, are used. Having established these conditions,single-base extension reactions are performed as well as 2-4 cycle SBSreactions with solution-based Raman detection using synthetic templatesand primers. The dsDNA extended with these NRTs generates the expectedRaman signal, which is eliminated upon cleavage with TCEP under aqueousconditions, and re-established with each round of SBS. Templatescomprising each of the complementary bases and repeated bases are usedto determine both SBS specificity and complete termination of thereaction by the NRTs. Washed aliquots taken after each step of the SBSreactions are collected, concentrated, and subjected to Raman detection.

(c) Surface-Enhanced Raman Scattering (SERS) Measurement:

Various methods of surface enhanced Raman scattering can increasesignals by up to 14 orders of magnitude.²⁹⁻³² Nucleotides are capturedon alumina nanotube porous surfaces containing aggregated metalnanoparticles (Au or Ag) to enhance the signals and measured by Ramanspectrometry following literature procedures (29-31). All four moleculesshould display a strong band at 2100 cm⁻¹. In contrast, dNTPs used ascontrols have no signal at this wavenumber.

(d) Solution-Based SBS with SERS Detection:

After testing the nucleotides themselves, single-base extensionreactions are conducted in solution, allowing specific incorporation ofeach of the four nucleotides, after which the extended DNA chains areadsorbed to the alumina-Ag or alumina-Au surfaces under non-denaturingconditions for SERS recording. In the same way, NRT-extended moleculestreated with TCEP are adsorbed to the surfaces and SERS measured. Thisis similar in principle to the solution-based assays for nucleotidesusing MALDI-TOF MS.

Producing SERS-Prepared Surfaces Designed Specifically for Detection ofthe Labeled Nucleotides

(a) Nanoplasmonic Antenna Arrays for SERS-SBS

In recent years, there have been several reports of significant Ramansignal enhancement at the single molecule level due to plasmoniccoupling to nearby nanostructures (33-35, 38-41). The work was doneusing roughened surfaces, metal films or discontinuous metal islandsdeposited on surfaces, colloidal powders, aqueous sols, and beads orscaffolds decorated with noble-metal colloids. These approaches hadlimited control over the nanostructure geometry and no control over thelocation of the analyte molecule relative to the plasmonic hot spots.More recent approaches have produced regularly ordered periodic particlearrays for generating promising SERS sensors. Li et al. used nanoimprintlithography (NIL) to form arrays of vertical posts which were coatedwith Au. The posts were coated with Au, and nanogaps formed on thesidewalls of the posts where traces of Au served as hot spots (42). Theyachieved impressively high area-average SERS enhancement of 1.2×10⁹ andgood large-area uniformity; this approach can be even more powerful ifone could control the formation of the metallic nanoislands on thesidewalls of the posts. A more controlled approach to localizedplasmonic enhancement uses bowtie nano-antennae (43-45). Nanoantennaecan enhance room-temperature fluorescence emission from a singlemolecule >1000 times more than molecules not coupled to the bowtie. Thisenhancement is the result of a roughly two-orders of magnitudeconcentration of the incident excitation field inside a ˜20 nm³ gap,together with an enhancement of the molecule's quantum yield by nearlyan order of magnitude due to the Purcell effect. The Purcell effectresults from an increased Rabi frequency, g, between the emitter and thebowtie optical mode, as shown in FIG. 11. This coupled bowtienanoantenna-molecule system forms an efficient interface betweenfar-field optics and molecular excited state and plasmonic excitations.

Described herein is a robust, solid-state platform for measuring theSERS signal resulting from the polymerase extension of specificoligonucleotides in SBS reactions. The platform integrates nanoplasmonicbowtie antennae together with site specific biomolecular interactions toyield reproducible and optimal signal enhancement. We combine plasmonicmodeling, advanced nanofabrication, selective biochemical surfacefunctionalization and photophysical analysis to design, build andmeasure arrays of nanoplasmonic antennae incorporating DNAfunctionalized sites. Initially, focus is on optimizing the SERS signalfrom few-molecule assemblies, to obtain robust, reproducible signalswith minimum scatter. Next, to reduce background from the pump laser byusing a new type of crossed-bowtie structure in which one pair oftriangles couples to the incident field, while the other pair couples tothe Raman scattered field in an orthogonal polarization. Thiscross-polarized design could enable SERS without the need for ahigh-resolution spectrometer or other spectral filters.

(b) Plasmonic Nanoantennae Design

To determine the optimal nanoantenna design, a variety of issues has tobe addressed: the molecule is deterministically positioned, using ananopositioning stage, to understand the position dependence ofsurface-enhanced Raman scattering near the bowtie gap. In similar recentexperiments with dielectric cavities, a single emitter was used to traceout the spatially resolved Purcell enhancement with 3-nm precision (46),as shown in FIG. 12. To maximize the Raman signal, bowtie resonatordesigns are optimized with resonances in the red, corresponding to theNRTs.

(c) Nanofabrication and Surface Functionalization

By combining advanced nanofabrication with selective biomolecularsurface functionalization, one could create functional surfaces withcontrol to the single-molecule level (47-53). Here, arrays of bowtienanoantennae with gap sizes ranging from ˜50 nm to 8 nm are fabricated.The surface is selectively functionalized within the gap to controlprimer concentration and thus the number of SBS reactions within eachgap, as shown in FIG. 11 a. The antenna structures are patterned byelectron beam lithography at 80-100 keV. In addition, resist processesallow one to reproducibly achieve sub-10 nm resolution (FIG. 11 b). Theprocesses rely on low temperature ultrasonic development and rinse oflow molecular weight PMMA, which prevents swelling and promotes highcontrast (54, 55). Ag or Al are deposited with a thin protective Ti capby electron beam evaporation. The bowties may be raised off thesubstrate surface by etching slightly into the substrate or depositing athin (few nm) spacer prior to metal deposition. After liftoff, thestructures are passivated with a thin CVD oxide layer, followed bydeposition of HMDS (hexamethyl-disilazane, which repels DNA). A secondlithography step defines primer adhesion sites, with PMMA once again asa resist. Following development, an O₂ reactive ion etch removes HMDS inthe patterned regions, exposing the oxide, rendering it hydrophilic,allowing the primer to adsorb only in the exposed area, thereby controlthe concentration and number of molecules within the gap. The process isshown in FIG. 13.

This design may be subject to the “moving target” problem, the situationwhere the Raman active NRTs extend beyond the optimal enhancement fieldas the hybridizing strand elongates. As one does not know a priori howsevere this problem is in practice, one can monitor the SERS signal as afunction of strand length. If the signal decays significantly, to modifythe surface functionalization to immobilize polymerase molecules withinthe bowtie gap using a process developed for controlling the placementof individual peptides and proteins on nanolithographically patternednanodots with dimensions as small as 4 nm or less (50, 56). A singlenanodot (or nanodot cluster) is lithographically patterned within thebowtie gap. DNA polymerase is bound to the nanodot, as shown in FIG. 14,using an amine-based linkage. This fixes the hybridization site towithin the optimal enhancement zone of the bowtie antenna, independentof the length of the DNA.

Another technique to reliably place a single primer molecule within aplasmonic hot spot with reliable control over all dimensions may be usedto achieve single-molecule probing. This technique relies on the use ofDNA origami (57). We have recently developed processes to place 5 nm Aunanoparticles at prescribed locations on origami scaffolds (FIG. 15 a)by integrating sticky end binding strands into programmed staplelocations within the origami framework to which nanoobjectsfunctionalized with complementary sticky ends can bind. This can placeobjects as close as 2 nm apart (58), and to place origami scaffolds atlithographically determined locations on a substrate using nanoimprintlithography (59), as shown in FIG. 15 b. Thus, construct arrays oforigami scaffolds upon which Au and Ag nanoparticles and nanorods areplaced at specified binding sites a few nanometers apart and position aprimer molecule between them is possible. This platform could provide areliable measure of SERS enhancement at the single molecule level.

Use of “plasmonic crystals”, arrayed plasmonic structures that arecoupled to show band formation, is contemplated, by employ periodicTiO-Au-TiO structures (FIG. 16) for enhanced light generation andextraction from InP-based quantum wells (60). The emission rateenhancement of molecules coupled to such structures could reach a factorof 50, more than what may be possible with bowtie antennae.

Experiments on spatially resolved plasmon-molecule coupling areconducted using scanning confocal microscopy setups. Using a set oftunable continuous-wave and picosecond pulsed lasers, operating in therange of 405-900 nm, one is able to optimally excite the plasmonicstructure, while positioning the molecule with nm resolution. Theseexperiments enable one to understand the physics governing the couplingbetween single emitters and localized and periodic plasmonic fieldconcentrators, providing the fundamental knowledge needed to developmore efficient plasmon-enhanced SERS devices. By applying techniquesfrom solid state cavity quantum electrodynamics (QED) to plasmonicnano-cavities (61-63) to efficiently pump and collect from singlemolecules coupled to the bowtie structures, a new range of quantumoptical interfaces for interacting with single molecules in acontrollable fashion can be developed.

Conducting Surface-Bound SBS Reactions with SERS Detection

Synthetic templates and PCR products are covalently attached toSERS-prepared surfaces, and hybridized with primers. Single-baseextension reactions are conducted with each of the four 3′-O—N₃-NRTs,adding them one by one. The overall scheme is shown in FIG. 17. In thisway, the incorporation specificity is measured by virtue of the Ramansignal. After cleavage and re-recording of the Raman spectrum, a secondand third cycle of SBS is conducted. Templates are designed to include 2of each base in a row, as a way of confirming that the NRTs arecompletely terminating the reactions. Lagging reactions due toincomplete removal of the azidomethyl groups are observed for.

There are several possibilities for attaching DNA to SERS surfaces. In anon-limiting example, the DNA is covalently linked to a carboxy-modifiedsilica slide using an NHS ester, after which the alumina and goldcoating is administered to the slide. Other chemistries are alsopossible, beginning with NH₂ or biotinylated surfaces. Depending on howmuch of the surface is coated, the opposite order of addition (SERScoating first, DNA attachment second) can be utilized. An alternativeapproach is to attach the DNA directly to silver nanoparticles (32).

A major advantage of the SERS-SBS approach is its high sensitivity andsimplicity. The dilution of samples to the limit of adequate andconsistent signal capture permits evaluation of the method. This permitsdirect mRNA sequencing, avoiding the biases inherent in cDNA synthesisand amplification in digital transcriptome analysis (RNA-Seq).

A strong Raman signal is obtained with SERS for individual NRTs, NRTsthat are part of DNA chains, and in surface-bound SBS reactions withthese NRTs. Of course, because the same band is obtained for each ofthese molecules, it is necessary to add the nucleotides one by one.

Design and Synthesis of Novel NRTs with 4 Distinct SERS Signatures toPerform SERS-SBS

To overcome the major limitation of the approach described above, i.e.the need to add each of the NRTs, one by one, due to their eachproducing an identical Raman peak, three additional nucleotides aresynthesized with distinct Raman signatures; thus providing a set of fourunique Raman tags, one for each of the four bases of DNA. There are twooptions. First, in the simplest and most elegant design, the moleculesare generated by attaching chemical groups with distinct SERS signaturesdirectly to the 3′-OH group. In this way, they serve at the same time asreversible termination groups and Raman tags. The NRTs in this categoryare depicted in FIG. 18, and a simplified synthetic scheme forgenerating such compounds is presented in FIG. 19. Each contains theazido peak at ˜2100 cm⁻¹, but in addition they display bands at2150-2250 depending on the additional groups included (cyano, alkyne,etc.). Most 3′-OH modifications in this size range are well accepted bythe mutant polymerases used for SBS with 3′-N₃-NRTs. Efficient cleavageof the blocking groups and their associated tags is accomplished withaqueous TCEP. The cleavage mechanism destroys the N₃ group and restoresthe 3′-OH, as shown in FIG. 20. Second, the new Raman signaling groupscan be attached to the base via an azidomethyl or other cleavable groupthat does not leave a chemical modification after cleavage. In thisvariation, it is preferred to have a separate reversible blocking groupon the 3′-OH. If necessary, a combination of both approaches can beutilized to achieve the desired set of four distinct Raman signatures asillustrated in FIG. 21. A synthetic scheme for the modified cytosineshown at the upper right in FIG. 21 is presented in FIG. 22. Allmolecules are characterized during and after synthesis by standardapproaches (MS, NMR, IR, SERS, etc.) to assess purity and product yield.

SBE reactions and cleavage in solution are conducted, and aliquots ofthe reaction mixture are adsorbed after incorporation and after cleavageto SERS-prepared surfaces for recording Raman spectra in the same way asdescribed above. However, in this case, all four NRTs can be added atthe same time, as indicated in FIG. 23, since the unique signature ofeach indicates the appropriate incorporation. Covalent attachment ofsynthetic templates to SERS-prepared surfaces, and hybridization ofprimers permits single-base extension reactions, with all four3′-O—N₃-NRTs. After cleavage and re-recording of the Raman spectrum, asecond and third cycle of SBS is performed. Templates include 2 of eachbase in a row, as a way of confirming that the NRTs are completelyterminating the reactions. Extensive cycles of SBS can be performed withSERS detection. DNA sample dilution is performed to quantify ultra-highsensitivity detection limits. Subsequently, bridge PCR is used toamplify a known DNA template on the SERS surface disclosed herein andthe 4-color Raman NRTs to test the read length and performance of theSERS-SBS system. The successful completion of this process lays a solidfoundation for the development of a routine SERS-SBS system.

Real-World Next-Generation Sequencing Libraries used to Compare SERS-SBS

Whole-genome sequencing can be obtained by first obtaining longsequencing reads on synthetic templates, and then sequencing a smalllibrary (e.g., a BAC shotgun library or a shotgun library derived from abacterial genome). DNA placed at several thousand positions per slide issufficient. In an embodiment, a thick layer with drilled wells can beused that can incorporate either DNA-attached beads, or wherein DNA isdirectly spotted, attached and amplified as necessary. These “masks” areattached to the surface of alumina nanotube-coated slides to which goldor silver particles are applied. In the simplest approach that avoidsissues of DNA attachment to the alumina-coated slides themselves, singlemolecules of DNA are amplified on beads by emulsion PCR and individualbeads are inserted into the drilled wells. The flow cell permitsadequate wash-through of reagents while not allowing the beads toescape. SBS is carried out under conditions established above. Methodsof attachment of DNA have been indicated above also.

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1. A nucleoside triphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, wherein R″ is OH or H, and wherein R′ is a hydrocarbyl, or asubstituted hydrocarbyl other than an azidomethyl, which has a Ramanspectroscopy peak with wavenumber from 2000 cm⁻¹ to 2300 cm⁻¹ or aFourier transform-infrared spectroscopy peak with wavenumber from 2000cm⁻¹ to 2300 cm⁻¹. 2-5. (canceled)
 6. The nucleoside triphosphateanalogue of claim 1, having a Raman spectroscopy peak with wavenumberfrom 2100 cm⁻¹ to 2260 cm⁻¹.
 7. A polynucleotide analogue, wherein thepolynucleotide analogue differs from a polynucleotide by comprising atits 3′ terminus one of the following structures in place of the H atomof the 3′ OH group normally present in the polynucleotide:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom.
 8. A composition comprising four different nucleoside triphosphate(NTP) analogues of claim 1, wherein R″ is H; and wherein R′ has thestructure:

wherein the wavy line indicates the point of attachment to the oxygenatom, and wherein (i) the structure of the R′ group of each of the fourNTP analogues is different from the structure of the R′ group of theremaining three NTP analogues, and (ii) each of the four NTP analoguescomprises a base which is different from the base of the remaining threeNTP analogues.
 9. A method for determining the sequence of consecutivenucleotide residues present in a single-stranded DNA comprising: (a)contacting the single-stranded DNA, having a primer hybridized to aportion thereof, with a DNA polymerase and four different nucleosidetriphosphate (NTP) analogues of claim 1 under conditions permitting theDNA polymerase to catalyze incorporation onto the primer of a NTPanalogue complementary to a nucleotide residue of the single-strandedDNA which is immediately 5′ to a nucleotide residue of thesingle-stranded DNA hybridized to the 3′ terminal nucleotide residue ofthe primer, so as to form a DNA extension product, wherein (i) each ofthe four NTP analogues has the structure:

wherein B is a base and is adenine, guanine, cytosine, or thymine, (ii)R′ has a predetermined Raman spectroscopy peak with wavenumber from 2000cm⁻¹ to 2300 cm⁻¹ and which is different from the wavenumber of theRaman spectroscopy peak of the other three NTP analogues, and (iii) eachof the four NTP analogues comprises a base which is different from thebase of the other three NTP analogues; (b) removing NTP analogues notincorporated into the DNA extension product; (c) determining after step(b) the wavenumber of the Raman spectroscopy peak of the NTP analogueincorporated in step (a) so as to thereby determine the identity of theincorporated NTP analogue and thus determine the identity of thecomplementary nucleotide residue in the single-stranded DNA; (d)treating the incorporated nucleotide analogue under specific conditionsso as to replace the R′ group thereof with an H atom thereby providing a3′ OH group at the 3′ terminal of the DNA extension product; and (e)iteratively performing steps (a) to (d) for each nucleotide residue ofthe single-stranded DNA to be sequenced except that in each repeat ofstep (a) the NTP analogue is (i) incorporated into the DNA extensionproduct resulting from a preceding iteration of step (a), and (ii)complementary to a nucleotide residue of the single-stranded DNA whichis immediately 5′ to a nucleotide residue of the single-stranded DNAhybridized to the 3′ terminal nucleotide residue of the DNA extensionproduct resulting from a preceding iteration of step (a), so as to forma subsequent DNA extension product, with the proviso that for the lastnucleotide residue to be sequenced step (d) is optional, therebydetermining the identity of each of the consecutive nucleotide residuesof the single-stranded DNA so as to thereby sequence the DNA.
 10. Amethod for determining the sequence of consecutive nucleotide residuesof a single-stranded DNA comprising: (a) contacting the single-strandedDNA, having a primer hybridized to a portion thereof, with a DNApolymerase and four different nucleoside triphosphate (NTP) analogues ofclaim 1 under conditions permitting the DNA polymerase to catalyzeincorporation into the primer of a NTP analogue complementary to anucleotide residue of the single-stranded DNA which is immediately 5′ toa nucleotide residue of the single-stranded DNA hybridized to the 3′terminal nucleotide residue of the primer, so as to form a DNA extensionproduct, wherein (i) each of the four NTP analogues has the structure:

wherein B is a base and is adenine, guanine, cytosine, or thymine, (ii)R′ has a predetermined Fourier transform-infrared spectroscopy peak withwavenumber from 2000 cm⁻¹ to 2300 cm⁻¹ and which is different from thewavenumber of the Fourier transform-infrared spectroscopy peak of theother three NTP analogues, and (iii) each of the four NTP analoguescomprises a base which is different from the base of the other three NTPanalogues; (b) removing NTP analogues not incorporated into the DNAextension product; (c) determining after step (b) the wavenumber of theFourier transform-infrared spectroscopy peak of the NTP analogueincorporated in step (a) so as to thereby determine the identity of theincorporated NTP analogue and thus determine the identity of thecomplementary nucleotide residue in the single-stranded DNA; (d)treating the incorporated nucleotide analogue so as to replace the R′group thereof with an H atom thereby providing a 3′ OH group at the 3′terminal of the DNA extension product; and (e) iteratively performingsteps (a) to (d) for each nucleotide residue of the single-stranded DNAto be sequenced except that in each repeat of step (a) the NTP analogueis (i) incorporated into the DNA extension product resulting from apreceding iteration of step (a), and (ii) complementary to a nucleotideresidue of the single-stranded DNA which is immediately 5′ to anucleotide residue of the single-stranded DNA hybridized to the 3′terminal nucleotide residue of the DNA extension product resulting froma preceding iteration of step (a), so as to form a subsequent DNAextension product, with the proviso that for the last nucleotide residueto be sequenced step (d) is optional, thereby determining the identityof each of the consecutive nucleotide residues of the single-strandedDNA so as to thereby sequence the DNA.
 11. (canceled)
 12. The method ofclaim 10, wherein the wavenumber of the Fourier transform-infraredspectroscopy peak is determined by irradiating the incorporated dNTPanalogue with grazing angle infra-red light.
 13. The method of claim 9,wherein the wavenumber of the Raman spectroscopy peak is determined byirradiating the incorporated dNTP analogue with 532 nm light. 14.(canceled)
 15. The method of claim 9, wherein the Raman spectroscopy issurface-enhanced Raman spectroscopy. 16-19. (canceled)
 20. A compositioncomprising four different nucleoside triphosphate (NTP) analogues ofclaim 1, wherein R″ is OH, wherein B is a base and is adenine, guanine,cytosine, or uracil, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, and wherein (i) the structure of the A′ group of each of the fourNTP analogues is different from the structure of the R′ group of theremaining three NTP analogues, and (ii) each of the four NTP analoguescomprises a base which is different from the base of the remaining threeNTP analogues.
 21. A nucleoside triphosphate analogue having thestructure:

wherein the base is adenine, guanine, cytosine, uracil or thymine,wherein R″ is an OH or an H, wherein L a cleavable linker, and wherein Rhas the structure:

wherein the wavy line indicates the point of attachment to L. 22-25.(canceled)
 26. A process for preparing a polynucleotide analogue ofclaim 7, comprising: contacting the polynucleotide with adeoxyribonucleoside triphosphate analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil orthymine, and wherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, in the presence of a DNA polymerase under conditions permittingincorporation of the deoxyribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between thedeoxyribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide. 27-28.(canceled)
 29. The process of claim 26, wherein the DNA polymerase is 9°N polymerase or a variant thereof, E. Coli DNA polymerase I,Bacteriophage T4 DNA polymerase, Sequenase, Tag DNA polymerase or 9° Npolymerase (exo-)A485L/Y409V.
 30. A process for preparing apolynucleotide analogue of claim 7 comprising: contacting thepolynucleotide with a ribonucleoside triphosphate analogue having thestructure:

wherein B is a base and is adenine, guanine, cytosine, or uracil, andwherein R′ has the structure:

wherein the wavy line indicates the point of attachment to the 3′ oxygenatom, in the presence of an RNA polymerase under conditions permittingincorporation of the ribonucleoside triphosphate analogue into thepolynucleotide by formation of a phosphodiester bond between theribonucleoside triphosphate analogue and a 3′ terminal of thepolynucleotide so as to thereby label the polynucleotide. 31-40.(canceled)
 41. A method for determining the identity of a nucleotideresidue within a stretch of consecutive nucleic acid residues in asingle-stranded DNA comprising: (a) contacting the single-stranded DNA,having a primer hybridized to a portion thereof such that the 3 terminalnucleotide residue of the primer is hybridized to a nucleotide residueof the single-stranded DNA immediately 3′ to the nucleotide residuebeing identified, with a DNA polymerase and at least four nucleosidetriphosphate (NTP) analogues of claim 1 under conditions permitting theDNA polymerase to catalyze incorporation into the primer of an NTPanalogue complementary to the nucleotide residue of the single-strandedDNA being identified, so as to form a DNA extension product, wherein (i)each of the four NTP analogues has the structure:

wherein B is a base and is adenine, guanine, cytosine, or thymine, (ii)R′ has a predetermined Raman spectroscopy peak with wavenumber which isfrom 2000 cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of theRaman spectroscopy peak of the other three NTP analogues or has apredetermined Fourier transform-infrared spectroscopy peak withwavenumber of from 2000 cm⁻¹ to 2300 cm⁻¹ and which is different fromthe wavenumber of the Fourier transform-infrared spectroscopy peak ofthe other three NTP analogues, and (iii) each of the four NTP analoguescomprises a base which is different from the base of the other three NTPanalogues; (b) removing NTP analogues not incorporated into the DNAextension product; and (c) determining the wavenumber of the Ramanspectroscopy peak or wavenumber of the Fourier transform-infraredspectroscopy peak of the NTP analogue incorporated in step (a) so as tothereby determine the identity of the incorporated NTP analogue and thusdetermine the identity of the complementary nucleotide residue in thesingle-stranded DNA, thereby identifying the nucleotide residue withinthe stretch of consecutive nucleic acid residues in the single-strandedDNA.
 42. A method for determining the identity of a nucleotide residuewithin a stretch of consecutive nucleic acid residues in asingle-stranded DNA comprising: (a) contacting the single-stranded DNA,having a primer hybridized to a portion thereof such that the 3 terminalnucleotide residue of the primer is hybridized to a nucleotide residueof the single-stranded DNA immediately 3′ to the nucleotide residueidentified, with a DNA polymerase and a nucleoside triphosphate (NTP)analogue of claim 1 under conditions permitting the DNA polymerase tocatalyze incorporation into the primer of the NTP analogue if it iscomplementary to the nucleotide residue of the single-stranded DNA beingidentified, so as to form a DNA extension product, wherein (i) the NTPanalogue has the structure:

wherein B is a base and is adenine, guanine, cytosine, or thymine, and(ii) R′ has a predetermined Raman spectroscopy peak with wavenumberwhich is from 2000 cm⁻¹ to 2300 cm⁻¹ or a predetermined Fouriertransform-infrared spectroscopy peak with wavenumber which is from 2000cm⁻¹ to 2300 cm⁻¹; (b) removing NTP analogues not incorporated into theDNA extension product; and (c) determining if the NTP analogue wasincorporated into the primer in step (a) by measuring after step (b) thewavenumber of the Raman spectroscopy peak or wavenumber of the Fouriertransform-infrared spectroscopy peak of any NTP analogue incorporated instep (a), wherein (1) if the NTP analogue was incorporated in step (a)determining from the wavenumber of the Raman spectroscopy peak orwavenumber of the Fourier transform-infrared spectroscopy peak measuredthe identity of the incorporated NTP analogue and thus determining theidentity of the complementary nucleotide residue in the single-strandedDNA, and (2) wherein if the NTP analogue was not incorporated in step(a) iteratively performing steps (a) through (c) until the complementarynucleotide residue in the single-stranded DNA is identified, with theproviso that each NTP analogue used to contact the single-stranded DNAtemplate in each subsequent iteration of step (a), (i) has apredetermined wavenumber of the Raman spectroscopy peak which isdifferent from the wavenumber of the Raman spectroscopy peak of everyNTP analogue used in preceding iterations of step (a) or has apredetermined Fourier transform-infrared spectroscopy peak which isdifferent from the Fourier transform-infrared spectroscopy peak of everyNTP analogue used in preceding iterations of step (a), and (ii)comprises a base which is different from the base of every NTP analogueused in preceding iterations of step (a), thereby identifying thenucleotide residue within the stretch of consecutive nucleic acidresidues in the single-stranded DNA.
 43. A method for determining theidentity of a nucleotide residue within a stretch of consecutive nucleicacid residues in a single-stranded DNA comprising: (a) contacting thesingle-stranded DNA with four different oligonucleotide probes, (1)wherein each of the oligonucleotide probes comprises (i) a portion thatis complementary to a portion of consecutive nucleotides of the singlestranded DNA immediately 3′ to the nucleotide residue being identified,and (ii) a 3′ terminal nucleotide residue analogue comprising on itssugar a 3′-O—R′ group wherein R′ is (a) is azidomethyl, or a substitutedor unsubstituted hydrocarbyl group and (b) has a predetermined Ramanspectroscopy peak with wavenumber which is from 2000 cm⁻¹ to 2300 cm⁻¹,and which is different from the wavenumber of the Raman spectroscopypeak of the R′ of the 3′ terminal nucleotide residue analogue of theother three oligonucleotide probes, or has a predeterminedFourier-transform infra red spectroscopy peak with wavenumber which isfrom 2000 cm⁻¹ to 2300 cm⁻¹, and is different from the wavenumber of theFourier-transform infra red spectra peak of the R′ of the 3′ terminalnucleotide residue analogue of the other three oligonucleotide probes,and (iii) each of the four terminal nucleotide residue analoguecomprises a base which is different from the base of the terminalnucleotide residue analogue of the other three oligonucleotide probes,and (2) under conditions permitting hybridization of the primer which isfully complementary to the portion of consecutive nucleotides of thesingle stranded DNA immediately 3′ to the nucleotide residue beingidentified; (b) removing oligonucleotide primers not hybridized to thesingle-stranded DNA; and (c) determining the wavenumber of the Ramanspectroscopy peak or wavenumber of the Fourier-transform infra red peakof the dNTP analogue of the oligonucleotide probe hybridized in step (a)so as to thereby determine the identity of the dNTP analogue of thehybridized oligonucleotide probe and thus determine the identity of thecomplementary nucleotide residue in the single-stranded DNA, therebyidentifying the nucleotide residue within the stretch of consecutivenucleic acid residues in the single-stranded DNA. 44-46. (canceled) 47.The method of claim 41, wherein the Raman spectroscopy issurface-enhanced Raman spectroscopy. 48-76. (canceled)
 77. The method ofclaim 42, wherein the Raman spectroscopy is surface-enhanced Ramanspectroscopy.
 78. The method of claim 43, wherein the Raman spectroscopyis surface-enhanced Raman spectroscopy.